Methods and compositions for preserving retinal ganglion cells

ABSTRACT

Provided are methods and compositions for maintaining the viability of retinal ganglion cells in a subject with an ocular disorder including, for example, glaucoma and optic nerve injury. The viability of the retinal ganglion cells can be preserved by administering a necrosis inhibitor either alone or in combination with an apoptosis inhibitor to a subject having an eye with the ocular condition. The compositions, when administered, maintain the viability of the cells and/or promote axon regeneration, thereby minimizing the loss of vision or visual function associated with the ocular disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/093,480, filed Apr. 7, 2016, which is a continuation of U.S. patentapplication Ser. No. 13/882,932, filed Sep. 30, 2013, which is thenational stage filing under 35 U.S.C. § 371 of International PatentApplication No. PCT/US2011/057327, filed Oct. 21, 2011, which claims thebenefit of and priority to U.S. Provisional Application No. 61/409,055,filed Nov. 1, 2010; U.S. Provisional Application No. 61/414,862, filedNov. 17, 2010; and U.S. Provisional Application No. 61/472,144, filedApr. 5, 2011; the disclosures of each application are herebyincorporated by reference in their entirety.

GOVERNMENT FUNDING

The work described in this application was sponsored, in part, by theNational Eye Institute under Grant No. EY14104. The United StatesGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The field of the invention relates generally to methods and compositionsfor preserving the viability of retinal ganglion cells (RGCs), forexample, in a subject affected with an ocular disorder wherein a symptomof the ocular disorder is loss of retinal ganglion cell viability. Moreparticularly, the invention relates to the use of a necrosis inhibitor,e.g., a RIP kinase inhibitor, e.g., a necrostatin, either alone or incombination with an apoptosis inhibitor, e.g., a pan-caspase inhibitor,for preserving the viability of retinal ganglion cells for the treatmentof the ocular disorder. The invention further relates to the use anecrosis inhibitor, either alone or in combination with an apoptosisinhibitor, for promoting axon regeneration with retinal ganglion cells.

BACKGROUND OF THE INVENTION

The retina is a delicate neural tissue lining the back of the eye thatconverts light stimuli into electric signals for processing by thebrain. The optic nerve is a cable of retinal ganglion cells that carrythe electric signals from the retina to the brain. Diseases affectingthe retina and optic nerve, including, for example, glaucoma, and opticnerve injury can lead to vision loss and blindness. Early detection andtreatment are critical in correcting problems before vision is lost inpreventing further deterioration of vision.

In the United States, glaucoma is the second leading cause of blindnessoverall. Glaucoma is a progressive disease which leads to optic nervedamage and, ultimately, total loss of vision. The causes of this diseasehave been the subject of extensive studies for many years, but are stillnot fully understood. The principal symptom of and/or risk factor forthe disease is elevated intraocular pressure or ocular hypertension dueto excess aqueous humor in the anterior chamber of the eye.Unfortunately, many of the drugs conventionally used to treat ocularhypertension have a variety of problems. For instance, miotics such aspilocarpine can cause blurring of vision and other visual side effects,which may lead either to decreased patient compliance or to terminationof therapy. Thus, there is a continuing need for therapies that controlelevated intraocular pressure associated with glaucoma without thedegree of undesirable side-effects attendant to most conventionaltherapies.

Damage to the optic nerve (ON) typically causes permanent andpotentially severe loss of vision. Like most pathways in the maturecentral nervous system, the optic nerve cannot regenerate if injured.Optic nerve injury can be the result of glaucoma, trauma, toxicity,inflammation, ischemia, congenital diseases, or compression from tumorsor aneurysms. To date, few effective treatments have been discovered torestore visual function and/or axon regeneration following optic nerveinjury.

Apoptosis and necrosis represent two different mechanisms of cell death.Apoptosis is a highly regulated process involving the caspase family ofcysteine proteases, and characterized by cellular shrinkage, chromatincondensation, and DNA degradation. In contrast, necrosis is associatedwith cellular and organelle swelling and plasma membrane rupture withensuing release of intracellular contents and secondary inflammation(Kroemer et al., (2009) CELL DEATH DIFFER 16:3-11). Necrosis has beenconsidered a passive, unregulated form of cell death; however, recentevidence indicates that some necrosis can be induced by regulated signaltransduction pathways such as those mediated by receptor interactingprotein (RIP) kinases, especially in conditions where caspases areinhibited or cannot be activated efficiently (Golstein P & Kroemer G(2007) TRENDS BIOCHEM. SCI. 32:37-43; Festjens et al. (2006) BIOCHIM.BIOPHYS. ACTA 1757:1371-1387). Stimulation of the Fas and TNFR family ofdeath domain receptors (DRs) is known to mediate apoptosis in most celltypes through the activation of the extrinsic caspase pathway. Inaddition, in certain cells deficient for caspase-8 or treated withpan-caspase inhibitor Z-VAD, stimulation of death domain receptors (DR)causes a RIP-1 kinase dependent programmed necrotic cell death insteadof apoptosis (Holler et al. (2000) NAT. IMMUNOL. 1:489-495; Degterev etal. (2008) NAT. CHEM. BIOL. 4:313-321). This novel mechanism of celldeath is termed “programmed necrosis” or “necroptosis” (Degterev et al.,(2005) NAT CHEM BIOL 1:112-119).

Receptor Interacting Protein kinase 1 (RIP-1) is a serine/threoninekinase that contains a death domain and forms a death signaling complexwith the Fas-associated death domain and caspase-8 in response to deathreceptor (DR) stimulation (Festjens et al. (2007) CELL DEATH DIFFER.14:400-410). During death domain receptor-induced apoptosis, RIP-1 iscleaved and inactivated by caspase-8, the process of which is preventedby caspase inhibition (Lin et al. (1999) GENES. DEV. 13:2514-2526). Ithas been unclear how RIP-1 kinase mediates programmed necrosis, butrecent studies revealed that the expression of RIP-3 and the RIP-1-RIP-3binding through the RIP homotypic interaction motif is a prerequisitefor RIP-1 kinase activation, leading to reactive oxygen species (ROS)production and necrotic cell death (He et al., (2009) CELL137:1100-1111; Cho et. al., (2009) CELL 137:1112-1123; Zhang et al.,(2009) SCIENCE 325:332-336).

There is still an ongoing need to minimize or eliminate cell death,e.g., retinal ganglion cell death, in certain ocular disorders, e.g., inglaucoma and optic nerve injury. It is contemplated that minimizingretinal ganglion cell death and/or promoting axon regeneration in theretinal ganglion cells will reduce the loss of vision or the loss ofvisual function associated with these various ocular disorders.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that a necrosisinhibitor, e.g., RIP kinase inhibitor, e.g., a necrostatin, e.g.,necrostatin-1, can be used to reduce or prevent the loss of retinalganglion cell viability, especially when the necrosis inhibitor iscombined with an apoptotic inhibitor (e.g., a pan-caspase inhibitor,e.g., Z-VAD and/or IDN-6556). It was previously understood that retinalganglion cell death associated with glaucoma and optic nerve injury wasprimarily caused by apoptosis. However, studies have shown that theadministration of Z-VAD, an apoptosis inhibitor (i.e., a pan-caspaseinhibitor), fails to thoroughly prevent retinal ganglion cell loss inglaucoma and optic nerve injury. The studies described hereinbelowindicate that, in the presence of an apoptosis inhibitor (e.g., apan-caspase inhibitor), retinal ganglion cells die by necrosis,including necroptosis (or programmed necrosis). These studies show thatprogrammed necrosis is a critical mechanism for ocular disorders whereina symptom of the disorder is the loss of retinal ganglion cell viabilityin the presence of a pan-caspase inhibitor. As a result, it is possibleto reduce the loss of visual function associated with an oculardisorder, in particular while the ocular disorder is being treated, byreducing the loss of retinal ganglion cell viability.

In one aspect, provided herein is a method preserving the visualfunction of an eye of a subject with an ocular condition, wherein asymptom of the ocular condition is the loss of retinal ganglion cellviability in the retina of the eye with the condition. The methodcomprises (a) administering to the eye of the subject an effectiveamount of a necrosis inhibitor and an effective amount of an apoptosisinhibitor thereby preserving the viability of the retinal ganglion cellsdisposed within the retina of the eye, and (b) then measuring the visualfunction (e.g., visual acuity) of the eye after the administration ofthe necrosis inhibitor and the apoptosis inhibitor. After administrationof the necrosis inhibitor and the apoptosis inhibitor the visualfunction (e.g., visual acuity) of the eye may be preserved or improvedrelative to the visual function of the eye prior to administration ofthe necrosis inhibitor and the apoptosis inhibitor. The ocular conditionmay include, but is not limited to, glaucoma, optic nerve injury, opticneuritis, optic neuropathies, central retinal artery occlusion, centralretinal vein occlusion and diabetic retinopathy. In an exemplaryembodiment, the ocular condition is glaucoma or optic nerve injury.

In another aspect, provided herein is a method of preserving theviability of retinal ganglion cells within the retina of a subject withan ocular condition. A symptom of the ocular condition may be the lossof retinal ganglion cells in the retina of the eye with the condition.The ocular condition may be glaucoma, optic nerve injury, opticneuritis, optic neuropathies, central retinal artery occlusion, centralretinal vein occlusion and diabetic retinopathy. The method comprisesadministering to the eye of the subject an effective amount of anecrosis inhibitor and an apoptosis inhibitor thereby preserving theviability of the retinal ganglion cells with the retina of the subjectwith the condition. After the administration of the necrosis inhibitorand the apoptosis inhibitor, the retinal ganglion cell is capable ofsupporting axonal regeneration.

In another aspect, provided herein is a method of preserving visualfunction of an eye of a subject affected with an ocular condition suchas glaucoma or optic nerve injury, wherein a symptom of the ocularcondition is loss of retinal ganglion cell viability, e.g., glaucoma,optic nerve injury, optic neuritis, optic neuropathies, central retinalartery occlusion, central retinal vein occlusion and diabeticretinopathy. The method comprises reducing the production and/oractivity of a RIP-1 kinase and/or RIP-3 kinase in the eye therebypreserving the viability of the retinal ganglion cells disposed with theretina of the eye. In certain embodiments, the reduction in theproduction or activity of the RIP-1 kinase and/or the RIP-3 kinase canbe achieved by administering an effective amount of RIP kinase (RIPK)inhibitor, e.g., a necrostatin.

In another aspect, provided herein is a method of preserving the visualfunction of an eye of a subject affected with an ocular conditionwherein a symptom of the ocular condition is loss of retinal ganglioncell viability in the retina of the eye. The method comprises (a)reducing the production or activity of a RIP-1 kinase and/or a RIP-3kinase in the eye thereby to preserve the viability of the retinalganglion cells disposed within the retina of the eye; and (b), aftertreatment, measuring visual function (e.g., visual acuity) of the eye.In certain embodiments, the reduction in the production or activity ofthe RIP-1 kinase and/or the RIP-3 kinase can be achieved byadministering an effective amount of RIPK inhibitor, e.g., anecrostatin. After administration of the RIPK inhibitor the visualfunction of the eye may be preserved or improved relative to the visualfunction of the eye prior to administration of the RIPK inhibitor.

In another aspect, the provided herein is a method for promoting axonalregeneration in an eye of a subject with an ocular condition, wherein asymptom of the ocular condition is the loss of retinal ganglion cellviability in the retina of an eye with the condition. The methodcomprises administering to the eye of the subject with the condition aneffective amount of a necrosis inhibitor and an effective amount of anapoptosis inhibitor thereby to promote the regeneration of a retinalganglion cell axon within the retina of the eye. The method may furthercomprise, after administration of the necrosis inhibitor and theapoptosis inhibitor, measuring visual function of the eye. Afteradministration of the necrosis inhibitor and the apoptosis inhibitor thevisual function of the eye may be preserved or improved relative to thevisual function of the eye prior to administration of the necrosisinhibitor and the apoptosis inhibitor. Visual function may be anindication of axon regeneration in the retinal ganglion cell. The ocularcondition may include, but is not limited to, glaucoma, optic nerveinjury, optic neuritis, optic neuropathies, central retinal arteryocclusion, central retinal vein occlusion and diabetic retinopathy.

In another aspect, provided herein is a combination of a necrosisinhibitor (e.g., a RIPK inhibitor, e.g., a necrostatin) and an apoptosisinhibitor (e.g., a pan-caspase inhibitor, e.g., Z-VAD or IDN-6556), foruse in preserving visual function of an eye of a subject affected withan ocular condition wherein a symptom of the ocular condition is loss ofretinal ganglion cell viability in the retina of the eye with thecondition. The ocular condition may be glaucoma, optic nerve injury,optic neuritis, optic neuropathies, central retinal artery occlusion,central retinal vein occlusion and diabetic retinopathy.

In another aspect, provided herein is a combination of a necrosisinhibitor (e.g., a RIPK inhibitor, e.g., a necrostatin) and an apoptosisinhibitor (e.g., a pan-caspase inhibitor, e.g., Z-VAD or IDN-6556), foruse in preserving the viability of retinal ganglion cells disposed inthe eye of a subject with an ocular condition, wherein a symptom of theocular condition is the loss of retinal ganglion cell viability in theretina of the eye with the condition. The ocular condition may beglaucoma, optic nerve injury, optic neuritis, optic neuropathies,central retinal artery occlusion, central retinal vein occlusion anddiabetic retinopathy.

In addition, provided herein is a combination of a necrosis inhibitor(e.g., a RIPK inhibitor, e.g., a necrostatin) and an apoptosis inhibitor(e.g., a pan-caspase inhibitor, e.g., Z-VAD or IDN-6556), for use inpromoting axon regeneration mediated via retinal ganglion cells in asubject with an ocular condition, for example, optic nerve injury.

In each of the foregoing aspects and methods, the necrosis inhibitor canbe a RIP kinase inhibitor, for example, a necrostatin. In certainembodiments of the foregoing methods, the necrostatin is necrostatin-1,necrostatin-2, necrostatin-3, necrostatin-4, necreostatin-5,necrostatin-7, or a combination thereof.

In certain embodiments, when a necrostatin is administered, thenecrostatin is administered to provide a final concentration ofnecrostatin in the eye greater than about 5 μM. For example, the finalconcentration of necrostatin in the eye may range from about 5 μM toabout 1000 μM, about 10 μM to about 1000 μM, about 100 μM to about 1000μM, about 150 μM to about 1000 μM, from about 200 μM to about 800 μM orfrom about 200 μM to about 600 μM. In certain embodiments, the finalconcentration of necrostatin in the eye is about 400 μM. In otherembodiments when a necrostatin is administered, from about 0.05 mg toabout 2 mg, 0.1 mg to about 1 mg, from about 0.2 mg to about 1 mg, orfrom about 0.2 mg to about 0.8 mg, of necrostatin can be administeredlocally to the eye of a mammal. In an exemplary embodiment, about 0.5 mgof necrostatin can be administered locally to the eye of a mammal.

In certain embodiments, when a pan-caspase inhibitor is administered,the pan-caspase inhibitor is administered to provide a finalconcentration of the pan-caspase inhibitor in eye greater than about 3μM. For example, the final concentration of pan-caspase inhibitor in theeye may range from about 3 μM to about 500 μM, about 10 μM to about 500μM, about 100 μM to about 500 μM, about 150 μM to about 500 μM, or fromabout 200 μM to about 400 μM. In certain embodiments, the finalconcentration of the pan-caspase inhibitor in the eye is about 300 μM.Exemplary pan-caspase inhibitors include zVAD, IDN-6556 or a combinationthereof. In other embodiments, from about 0.05 mg to about 1.5 mg, fromabout 0.15 mg to about 1.5 mg, from about 0.2 mg to about 1 mg, fromabout 0.2 mg to about 0.8 mg, from about 0.4 mg to about 1 mg, or fromabout 0.5 mg to about 0.8 mg, of the pan-caspase inhibitor can beadministered locally to the eye of a mammal. In an exemplary embodiment,about 0.7 mg of a pan-caspase inhibitor can be administered locally tothe eye of a mammal.

The necrosis inhibitor, e.g., a necrostatin, and/or the apoptosisinhibitor may be administered to the eye by intraocular, intravitreal,subretinal or trasscleral administration. The necrosis inhibitor, e.g.,a necrostatin, and/or the apoptosis inhibitor may be solubilized in aviscoelastic carrier that is introduced into the eye. In otherembodiments, the necrosis inhibitor, e.g., a necrostatin, and/or theapoptosis inhibitor may be administered systemically.

It is understood that the necrosis inhibitor, e.g., a necrostatin,and/or the apoptosis inhibitor may be administered sequentially orsimultaneously. The necrosis inhibitor, e.g., a necrostatin, and theapoptosis inhibitor may be administered in the same or differentcarriers.

In each of the foregoing methods and compositions, the necrostatin canbe selected from one or more of the following necrostatins. For example,in certain embodiments, the necrostatin is a Nec-1 related compound ofFormula I:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein

X is O or S;

R₁ is hydrogen, C₁-C₆alkyl, C₁-C₆alkoxyl, or halogen; and

R₂ is hydrogen or C₁-C₆alkyl.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-1 related compound of Formula I-A:

or a pharmaceutically acceptable salt, ester, or prodrug thereof, oroptical isomers or racemic mixtures thereof, wherein R₁ is H, alkyl,alkoxyl, or a halogen and R₂ is H or an alkyl.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-1 related compound of Formula I-B:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-1 related compound of Formula I-C:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-1 related compound of Formula I-D:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-1 related compound of Formula I-E:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein R₁ is H, alkyl, alkoxyl, or a halogen (for example, F, Cl, Br orI) and R₂ is H or an alkyl.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-1 related compound of Formula I-F:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-1 related compound of Formula I-G:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-2 related compound of Formula II:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

X is —CH₂—, —C(H)(R₁₄)—, —C(═S)—, —C(═NH)—, or —C(O)—;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, and R₁₀ each represent independentlyhydrogen, acyl, acetyl, alkyl, halogen, amino, C₁-C₆alkoxyl, nitro,—C(O)R₁₂, —C(S)R₁₂, —C(O)OR₁₂, —C(O)NR₁₂R₁₃, —C(S)NR₁₂R₁₃, or —S(O₂)R₁₂;

R₁₁ is hydrogen, acyl, acetyl, alkyl, or acylamino;

R₁₂ and R₁₃ each represent independently hydrogen, an optionallysubstituted alkyl, an optionally substituted aryl, an optionallysubstituted heteroaryl, an optionally substituted aralkyl, or anoptionally substituted heteroaralkyl;

R₁₄ is acyl, acetyl, alkyl, halogen, amino, acylamino, nitro, —SR₁₁,—N(R₁₁)₂, or —OR₁₁;

the bond indicated by (a) can be a single or double bond; and

the bond indicated by (b) can be a single or double bond.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-2 related compound of Formula IIA:

or a pharmaceutically acceptable salt thereof, wherein:

R₁, R₂, R₅, R₆, R₇, and R₁₀ each represent independently hydrogen,alkyl, halogen, amino, or methoxyl; and

R₃, R₄, R₈, and R₉ are C₁-C₆alkoxyl.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-3 related compound of Formula III:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

Z is —CH₂—, —CH₂CH₂—, —O—, —S—, —S(O)—, —S(O₂)—, or —N(R₇)—;

R₁, R₃, and R₅ each represent independently for each occurrencehydrogen, halogen, hydroxyl, amino, C₁-C₆alkyl, C₁-C₆alkoxy,C₁-C₆alkoxy-C₁-C₆alkyl, C₁-C₆alkanoyl, C₁-C₆alkylsulfinyl,C₁-C₆alkylsulfinyl-C₁-C₆alkyl, C₁-C₆alkylsulfonyl,C₁-C₆alkylsulfonyl-C₁-C₆alkyl, aryl, aralkyl, heterocycloalkyl,heteroaryl, or heteroaralkyl;

R₂ and R₄ are C₁-C₆alkoxy;

R₆ is —C(O)R₈, —C(S)R₈, —C(O)OR₈, —C(O)NR₈R₉, —C(S)NR₈R₉, —C(NH)R₈, or—S(O₂)R₈;

R₇ is alkyl, aralkyl, or heteroaralkyl;

R₈ and R₉ each represent independently hydrogen, C₁-C₆alkyl,heteroalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

n represents independently for each occurrence 0, 1, or 2.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-4 related compound of Formula IV:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

R₁ is

R₂ and R₃ each represent independently for each occurrence hydrogen ormethyl;

R₄ represents independently for each occurrence halogen, hydrogen,C₁-C₆alkyl, C₂-C₆alkenyl, or C₂-C₄alkynyl;

R₅ is C₁-C₄alkyl;

R₆ is hydrogen, halogen, or —CN;

R₇ is hydrogen or C₁-C₄alkyl;

R₈ is C₁-C₆alkyl, or R₈ taken together with R₉, when present, forms acarbocyclic ring;

R₉ is hydrogen or C₁-C₆alkyl, or R₉ taken together with R₈ forms acarbocyclic ring;

R₁₀ is hydrogen or C₁-C₆alkyl;

A is phenylene or a 5-6 membered heteroarylene;

X is N or —C(R₉)—;

Y is N or —C(R₁₀)—;

Z is S or O; and

m and n each represent independently 1, 2, or 3.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-4 related compound of Formula IV-A:

or a pharmaceutically acceptable salt thereof.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-5 related compound of Formula V:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

A is a saturated or unsaturated 5-6 membered carbocyclic ring;

X is a bond or C₁-C₄alkylene;

R₁ is C₁-C₆ alkyl, halogen, hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄,CO₂R₄, or C(O)N(R₄)₂;

R₂ is

R₃ is —C₁-C₆alkylene-CN, —CN, C₁-C₆alkyl, or C₂-C₆alkenyl;

R₄ represents independently for each occurrence hydrogen, C₁-C₆alkyl,aryl, or aralkyl;

R₅ represents independently for each occurrence C₁-C₆alkyl, halogen,hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄, CO₂R₄, or C(O)N(R₄)₂;

B is a 5-6 membered heterocyclic or carbocylic ring; and

n and p each represent independently 0, 1, or 2.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-5 related compound of Formula V-A:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

R₁ is C₁-C₆alkyl, halogen, hydroxyl, C₁-C₆alkoxyl, or —N(R₄)₂;

R₂ is

R₃ is —C₁-C₆alkylene-CN;

R₄ represents independently for each occurrence hydrogen, C₁-C₆alkyl,aryl, or aralkyl;

R₅ represents independently for each occurrence C₁-C₆alkyl, halogen,hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄, CO₂R₄, or C(O)N(R₄)₂;

B is a 5-6 membered heterocyclic or carbocylic ring; and

n and p each represent independently 0, 1, or 2.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-7 related compound of Formula VII:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

R₁, R₂, and R₃ each represent independently hydrogen or C₁-C₄alkyl;

R₄ is

R₅ and R₆ each represent independently for each occurrence halogen,C₁-C₆alkyl, hydroxyl, C₁-C₆alkoxyl, —N(R₇)₂, —NO₂, —S—C₁-C₆alkyl,—S-aryl, —SO₂—C₁-C₆alkyl, —SO₂-aryl, —C(O)R₇, —CO₂R₇, —C(O)N(R₇)₂,heterocycloalkyl, aryl, or heteroaryl;

R₇ represents independently for each occurrence hydrogen, C₁-C₆alkyl,aryl, or aralkyl; or two occurrences of R₇ attached to the same nitrogenatom are taken together with the nitrogen atom to which they areattached to form a 3-7 membered heterocyclic ring;

A is a 5-6 membered heterocyclic ring; and

p is 0, 1, or 2.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-7 related compound of Formula VIII:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

each X¹, X², X³, X⁴, X⁵, and X⁶ is selected, independently, from N orCR^(X1);

each Y¹, Y², and Y³ is selected, independently, from O, S, NR^(Y1), orCR^(Y2)R^(Y3);

each Z¹ and Z² is selected, independently, from O, S, or NR^(Z1);

each R^(Y1) and R^(Z1) is selected, independently, from H, optionallysubstituted C₁₋₆alkyl, optionally substituted C₂₋₆alkenyl, optionallysubstituted C₂₋₆alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(5A), —C(═O)OR^(5A), or—C(═O)NR^(5A)R^(6A);

each R^(X1), R^(Y2), and R^(Y3) is selected, independently, from H,halogen, CN, NC, NO₂, N₃, OR³, SR³, NR³R⁴, —C(═O)R^(5A), —C(═O)OR^(5A),—C(═O)NR^(5A)R^(6A), —S(═O)R^(5A), —S(═O)₂OR^(5A), —S(═O)₂OR^(5A),—S(═O)₂OR^(5A)R^(6A), optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkenyl, optionally substituted C₂₋₆alkynyl, optionallysubstituted cycloalkyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl;

each R¹, R² R^(5A), R^(5B), R^(6A), and R^(6B) is selected from H,optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆alkenyl,optionally substituted C₂₋₆alkynyl, optionally substituted cycloalkyl,optionally substituted heterocyclyl, optionally substituted aryl, oroptionally substituted heteroaryl; or R^(5A) and R^(6A), or R^(5B) andR^(6B) combine to form a heterocyclyl; and

each R³ and R⁴ is selected from H, optionally substituted C₁₋₆ alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,—C(═O)R^(5B), —C(═S)R^(5B), —C(═NR^(6B))R^(5B), —C(═O)OR^(5B),—C(═O)NR^(5B)R^(6B), —S(═O)R^(5B), —S(═O)₂R^(5B), —S(═O)₂OR^(5B), or—S(═O)₂NR^(5B)R^(6B). In certain embodiments when R¹ is H, X¹, X², andX⁴ are each CH, X³, X⁵, and X⁶ are each N, Y¹ and Y³ are each S, Y² isNH, Z¹ is NH, and Z² is O, then R² is not 4-fluorophenyl.

In each of the foregoing methods and compositions, the necrostatin canbe a Nec-4 related compound of Formula IX:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

X₁ and X₂ are, independently, N or CR⁴;

X₃ is selected from O, S, NR⁵, or —(CR⁵)₂;

Y is selected from C(O) or CH₂; and

Z is (CR⁶R⁷)_(n);

R¹ is selected from H, halogen, optionally substituted C₁₋₆alkyl, oroptionally substituted C₁₋₆cycloalkyl, or optionally substituted aryl;

R² is selected from H or optionally substituted C₁₋₆alkyl;

R³ is optionally substituted aryl;

each R⁴ is selected from H, halogen, carboxamido, nitro, cyano,optionally substituted C₁₋₆alkyl, or optionally substituted aryl;

R⁵ is selected from H, halogen, optionally substituted C₁₋₆alkyl, oroptionally substituted aryl;

each R⁶ and R⁷ is, independently, selected from H, optionallysubstituted C₁₋₆alkyl, or aryl; and

n is 0, 1, 2, or 3. In certain embodiments, when X₁ and X₂ are N, X₃ isS, Y is C(O), Z is CH₂, R² is H, and R³ is 2-chloro-6-fluoro-phenyl,then R¹ is not methyl.

The foregoing aspects and embodiments of the invention may be more fullyunderstood by reference to the following figures, detailed descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention may be more fully understoodby reference to the drawings described herein.

FIG. 1 provides a schematic diagram of retinal ganglion cell death.

FIGS. 2A-2D provide graphs and a photograph showing TNF-α expression(FIG. 2A and FIG. 2B), RGC numbers per field (FIG. 2C), and IPLthickness (FIG. 2D) in mice that underwent ON injury and were injectedwith an anti-TNF-α neutralizing antibody. FIGS. 2E-2F provide graphsshowing RIP1 and RIP3 expression in the retina of mice that underwent ONinjury. FIG. 2G provides a photograph and FIGS. 2I and 2H, respectively,provide graphs showing RIP1 and RIP3 protein levels in the retina ofmice having undergone ON injury.

FIGS. 3A-3G provide photographs and graphs showing TUNEL-positive cells(FIG. 3A and FIG. 3B), Brn3B-positive cells (FIG. 3C and FIG. 3D), IPLthickness (FIG. 3E), GCL and IPL thickness (FIG. 3F and FIG. 3G) inZ-VAD and/or Nec-1 treated mice that underwent ON injury at day one.FIGS. 3H-3J provide photographs and graphs showing caspase-8 (FIG. 3H),caspase-9 (FIG. 3I and FIG. 3K) and caspase-3 (FIG. 3J and FIG. 3K)expression in Z-VAD and/or Nec-1 treated mice that underwent ON injuryone day after injury. FIG. 3L provide a graph showing TNF-α expressionin Z-VAD and/or Nec-1 treated mice that underwent ON injury one dayafter ON injury. FIGS. 3M-3Q provide photographs and graphs showingTUNEL-positive cells (FIG. 3M and FIG. 3N), Brn3B-positive cells (FIG.3O and FIG. 3P), and IPL thickness (FIG. 3Q) in Z-VAD and/or Nec-1treated mice that underwent ON injury at day one, day three, or day 7after ON injury. FIG. 3R provides a graph showing caspase-3, caspase-8,and caspase-9 activities in the retina one day after ON injury (n=6,*p<0.01). The caspase activities were normalized to non-injured retina.

FIGS. 4A-4B provide a photograph of PI staining (FIG. 4A) and graph(FIG. 4B) showing quantification of PI-positive cells in ZVAD and/orNec-1 treated mice that underwent ON injury. FIGS. 4C-4D provide (FIG.4C) TEM photographs of RGCs one day after ON injury and (FIG. 4D)quantification of apoptotic and necrotic RGC death.

FIGS. 5A-5G provide photographs and graphs showing TUNEL-positive cells(FIG. 5A and FIG. 5B), Brn3B-positive cells (FIG. 5C and FIG. 5D), IPLthickness (FIG. 5E), and PI-positive cells (FIG. 5F and FIG. 5G) inZ-VAD and/or Nec-1 treated RIP3−/− mice that underwent ON injury.

FIGS. 6A-6H provide photographs and graphs showing TNF-α mRNA level(FIG. 6A; n=6, *p<0.01) and RIP3 and RIP1 mRNA and protein levels (FIG.6B, FIG. 6C, and FIG. 6D; n=6, *p<0.01, **p<0.05), TUNEL-positive cells(FIG. 6E and FIG. 6F; n=8, *p<0.01), Brn3B-positive cells (FIG. 6G; n=8,**p<0.05), and IPL thickness (FIG. 6H), in RIP3−/− mice that underwentNMDA-induced ON injury. Bars for FIG. 6E and FIG. 6G are 100 μm. GCL;retinal ganglion cell layer, INL; inner nuclear layer, ONL; outernuclear layer.

FIGS. 7A-7B provide a photograph and graph showing AIF translocationfollowing Z-VAD and/or Nec-1 treatment in wildtype or RIP3−/− mice. FIG.7C provides a graph showing ROS production in wildtype or RIP3−/− mice.

FIG. 8A-8B provide transmission electron microscope (TEM)photomicrographs depicting RGCs after ON injury. FIG. 8B indicatesformation of autophagosomes (black arrows) and autolysosome (blackarrowheads) in necrotic cells with cellular swelling (left and middlepanel) and swollen axons (right panel).

FIGS. 9A-9C provide graphs showing Atg5 (FIG. 9A), Atg7 (FIG. 9B), andAtg12 (FIG. 9C) expression after ON injury, as determined byquantitative real-time PCR analysis. FIGS. 9D-9E provide photographsshowing LC3 protein levels and immunostaining at one day after ONinjury.

FIGS. 10A-10D provide photographs and a graph showing TUNEL-positivecells (FIG. 10A and FIG. 10B) and Brn3b-positive cells (FIG. 10C andFIG. 10D) in mice that were treated with 3-MA. FIGS. 10E-10F providegraphs depicting IPL thickness (FIG. 10E) and carbonyl contents in micethat were treated with 3-MA (FIG. 10F).

FIG. 11 provides a graph showing RGC survival in mice that were treatedwith Z-VAD and/or Nec-1 following optic nerve crush injury.

FIGS. 12A-12E provide photographs showing axon regeneration in micetreated with Z-VAD and/or Nec-1 following optic nerve crush injury.FIGS. 12A-12E show longitudinal sections of the optic nerve stained withan antibody against βIII-tubulin, following optic nerve crush injury.The vertical arrows denote the locations of the injury sites, and thehorizontal reference lines denote regions where axon regeneration weredetected following treatment with Nec-1 and ZVAD (FIGS. 12D-12E versusFIGS. 12A, 12B, and 12C).

DETAILED DESCRIPTION

The invention relates to methods and composition for preserving theviability of retinal ganglion cells disposed within a retina of an eyeof a subject with certain ocular condition, e.g., glaucoma, optic nerveinjury, optic neuritis, optic neuropathies, diabetic retinopathy,central retinal artery occlusion, and central retinal vein occulusion,wherein the viability of the retinal ganglion cells are affected by theocular condition. Using the methods and compositions described herein,it may be possible to preserve or improve visual function in the eye bymaintaining retinal ganglion cell viability while the underlying ocularcondition is being treated. The methods and compositions describedherein may further promote axon regeneration in the retinal ganglioncells and reduce the loss of vision or the loss of visual functionassociated with the various ocular conditions.

As demonstrated herein, programmed necrosis appears to be a criticalmechanism of retinal ganglion cell death in certain ocular conditions,for example, glaucoma or optic nerve injury in the presence of anapoptosis inhibitor, e.g., a pan-caspase inhibitor. As depicted in FIG.1, there are two pathways for cell death (apoptosis and necrosis), whichappear to be mediated by RIP-1, a serine/threonine kinase. RIP1 forms adeath inducing signaling complex with Fas-associated domain (FADD) andcaspase-8, thereby activating caspase-8 and the downstream cascadeleading to apoptosis. On the other hand, when caspase pathway is blocked(for example, with a caspase inhibitor such as ZVAD), RIP1 kinase isactivated in a RIP1-RIP3 complex and promotes RGC necrosis. Althoughautophagy is also activated during RGC death, it is mainly associatedwith necrotic RGC death. Thus, RIP kinases act as common intermediariesfor various upstream death signals, and their blockade in addition tocaspases is likely necessary for effective neuroprotection.

The methods and compositions described herein are directed to therapiesthat target both the necrotic and apoptotic pathways of programmed celldeath. In particular, the methods and compositions disclosed hereinfacilitate a combination therapy where a necrosis inhibitor, e.g., anecrostatin (e.g., necrostatin-1 or necrostatin-4), can be administeredeither alone or in combination (either sequentially or simultaneously)with an apoptosis inhibitor e.g., a pan-caspase inhibitor (e.g., Z-VADor IDN-6556). In certain embodiments, the disclosed methods surprisinglyuse necrostatins at concentrations higher than those previously thoughtto be clinically tolerable. Moreover, it has been demonstrated that thecombination of a necrostatin, e.g., necrostatin-1 or necrostatin-4, anda pan-caspase inhibitor, e.g., Z-VAD or IDN-6556, produce a superioreffect in reducing retinal ganglion cell death, compared to either drugalone. It has been further demonstrated that the combination treatmentof a necrostain and a pan-caspase inhibitor promotes axon regenerationin the retinal ganglion cells following optic nerve injury.

For convenience, certain terms in the specification, examples, andappended claims are collected in this section.

As used herein, the term “cell death” is understood to mean the death ofa cell, e.g., by apoptosis or necrosis.

As used herein, the term “apoptosis” is understood to meancaspase-dependent cell death, which is characterized by any of thefollowing properties: cell shrinkage, nuclear condensation, DNAfragmentation or membrane blebbing.

As used herein, the term “apoptosis inhibitor” is understood to mean anyagent that, when administered to a mammal, reduces apoptotic cell deathin retinal ganglion cells. For example, it is understood that certainuseful apoptosis inhibitors act by reducing or eliminating the activityof one or more members of the intrinsic or extrinsic or common apoptoticpathways. Furthermore, it is understood that an agent that eitherdirectly or indirectly affects the activity of one or more caspases(e.g., a pan-caspase inhibitor) is considered to be an apoptosisinhibitor. It is understood that a caspase inhibitor can affect theactivity of a caspase either directly by modulating a specific caspasein the apoptotic pathway or indirectly by modulating a downstreamcaspase present in the apoptotic pathway.

As used herein, the term “pan-caspase inhibitor” is understood to mean abroad-spectrum caspase inhibitor that inhibits at least two, preferablyat least three different caspases (e.g., caspase-1, caspase-2,caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8,caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and/orcaspase-14. Z-VAD (also known asBenzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone andcarbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) is anexemplary pan-caspase inhibitor and is available from R&D Systems (Cat.No. FMK001) and Promega (Cat. No. G7231). Other exemplary pan-caspaseinhibitors that may be used include IDN-6556 (also known as“PF-3,491,390”) available from Conatus Pharmaceuticals, Inc. (formerlyIdun Pharmaceuticals, Inc.), VX-799 available from VertexPharmaceuticals, Inc., MX1013 available Maxim Pharmaceuticals, Inc.,Xyz033mp available from LG Chemical, Inc., all of which are described,for example, in Linton, S. D. (2005) CURRENT TOPICS IN MEDICAL CHEM.5:1697-1717. It is understood that a “pan-caspase inhibitor” may also bea cocktail (e.g., a combination) of caspase inhibitors including two ormore of specific caspase inhibitors (e.g., synthetic or endogenouscaspase inhibitors).

As used herein, the term “necrosis” is understood to meancaspase-independent cell death characterized by any of the followingproperties: cellular and/or organelle swelling, plasma membrane rupture,or discontinuity in plasma, nuclear and/or organelle membranes. As usedherein, the terms “necroptosis” and “programmed necrosis” refer to aform of necrosis and is understood to mean one form of programmed orregulated necrosis, and in certain embodiments, necroptosis is mediatedby the serine/threonine kinase activity of receptor interacting protein(RIP) kinases, for example, RIP-1 kinase and/or RIP-3 kinase.

As used herein, the term “necrosis inhibitor” is understood to mean anagent, which, when administered to a mammal, reduces necrotic cell deathin retinal ganglion cells. For example, it is understood that certainnecrosis inhibitors act by reducing or inhibiting necroptosis orprogrammed necrosis. A necrosis inhibitor can be an agent that modulatesthe production and/or activity of one or more RIP kinases (e.g., RIP-1kinase and/or RIP-3 kinase). For example, an inhibitor of RIP-1 kinaseis understood to modulate the activity of RIP-1 kinase as well asdownstream RIP kinases, e.g., RIP-3 kinase, in the necrosis cascade.Accordingly, a RIP-1 kinase inhibitor is also understood to modulateRIP-3 kinase activity.

As used herein, the term “necrostatin” or “nec” is understood to mean aninhibitor of caspase-independent cell death or necroptosis. Exemplarynecrostatins include necrostatin-1 (“Nec-1”), necrostatin-2 (“Nec-2”),necrostatin-3 (“Nec-3”), necrostatin-4 (“Nec-4”), necrostatin-5(“Nec-5”) and necrostatin-7 (“Nec-7”).

In certain embodiments, the necrostatin is a Nec-1 related compound ofFormula I:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein

X is O or S;

R₁ is hydrogen, C₁-C₆alkyl, C₁-C₆alkoxyl, or halogen; and

R₂ is hydrogen or C₁-C₆alkyl.

In certain embodiments, X is O. In certain embodiments, R₁ is hydrogenor halogen (such as chlorine). In certain embodiments, R₂ is a methyl orethyl. In certain other embodiments, R₁ is hydrogen or Cl, and R₂ is amethyl.

In certain embodiments, the necrostatin is a Nec-1 related compound ofFormula I-A, shown below:

or a pharmaceutically acceptable salt, ester, or prodrug thereof, oroptical isomers or racemic mixtures thereof, wherein R₁ is H, alkyl,alkoxyl, or a halogen (for example, F, Cl, Br or I) and R₂ is H or analkyl. In certain embodiments, R₁ is H or Cl. In certain otherembodiments, R₂ is a methyl or ethyl. In certain other embodiments, R₁is H or Cl, and R₂ is a methyl.

In certain other embodiments, the necrostatin is a Nec-1 relatedcompound of Formula I-B, shown below:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In certain other embodiments, the necrostatin is a Nec-1 relatedcompound of Formula I-C, shown below:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In certain other embodiments, the necrostatin is a Nec-1 relatedcompound of Formula I-D, shown below:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In certain other embodiments, the necrostatin is a Nec-1 relatedcompound of Formula I-E, shown below:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein R₁ is H, alkyl, alkoxyl, or a halogen (for example, F, Cl, Br orI) and R₂ is H or an alkyl. In certain embodiments, R₁ is H or Cl. Incertain other embodiments, R₂ is a methyl or ethyl. In certain otherembodiments, R₁ is H or Cl, and R₂ is a methyl.

In certain other embodiments, the necrostatin is a Nec-1 relatedcompound of Formula I-F, shown below:

or a pharmaceutically acceptable salt, ester, or prodrug thereof. Incertain other embodiments, the necrostatin is a Nec-1 related compoundof Formula I-G, shown below:

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

The Nec-1 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as in Degterev etal., (2005) NAT CHEM BIOL 1:112-119; Degterev et al., (2008) NAT CHEMBIOL 4:313-321; and International Patent Application Publication No. WO2007/075772, all of which are hereby incorporated by reference.

In certain embodiments, the necrostatin is a Nec-2 related compound ofFormula II:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

X is —CH₂—, —C(H)(R₁₄)—, —C(═S)—, —C(═NH)—, or —C(O)—;

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, and R₁₀ each represent independentlyhydrogen, acyl, acetyl, alkyl, halogen, amino, C₁-C₆alkoxyl, nitro,—C(O)R₁₂, —C(S)R₁₂, —C(O)OR₁₂, —C(O)NR₁₂R₁₃, —C(S)NR₁₂R₁₃, or —S(O₂)R₁₂;

R₁₁ is hydrogen, acyl, acetyl, alkyl, or acylamino;

R₁₂ and R₁₃ each represent independently hydrogen, an optionallysubstituted alkyl, an optionally substituted aryl, an optionallysubstituted heteroaryl, an optionally substituted aralkyl, or anoptionally substituted heteroaralkyl;

R₁₄ is acyl, acetyl, alkyl, halogen, amino, acylamino, nitro, —SR₁₁,—N(R₁₁)₂, or —OR₁₁;

the bond indicated by (a) can be a single or double bond; and

the bond indicated by (b) can be a single or double bond.

In certain embodiments, X is —C(O)—. In certain embodiments, R₁, R₂, R₅,R₆, R₇, and R₁₀ each represent independently hydrogen, acyl, alkyl,halogen, or amino. In certain embodiments, R₃, R₄, R₈, and R₉ areC₁-C₆alkoxyl. In certain embodiments, the bond indicated by (a) is adouble bond; and the bond indicated by (b) is a double bond. In certainembodiments, when each of R₁, R₄, R₅, R₆, R₉ and R₁₀ is hydrogen andeach of R₂, R₃, R₇, and R₈ is methoxyl, then X is not —C(O)—, —CH₂—, or—CH(OH)—.

In certain other embodiments, the necrostatin is a Nec-2 relatedcompound of Formula II-A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁, R₂, R₅, R₆, R₇, and R₁₀ each represent independently hydrogen,alkyl, halogen, amino, or methoxyl; and

R₃, R₄, R₈, and R₉ are C₁-C₆alkoxyl.

In certain other embodiments, the Nec-2 related compound is

or a pharmaceutically acceptable salt thereof.

The Nec-2 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as inInternational Patent Application Publication No. WO 2007/075772, whichis hereby incorporated by reference.

In certain embodiments, the necrostatin is a Nec-3 related compound ofFormula III:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

Z is —CH₂—, —CH₂CH₂—, —O—, —S—, —S(O)—, —S(O₂)—, or —N(R₇)—;

R₁, R₃, and R₅ each represent independently for each occurrencehydrogen, halogen, hydroxyl, amino, C₁-C₆alkyl, C₁-C₆alkoxy,C₁-C₆alkoxy-C₁-C₆alkyl, C₁-C₆alkanoyl, C₁-C₆alkylsulfinyl,C₁-C₆alkylsulfinyl-C₁-C₆alkyl, C₁-C₆alkylsulfonyl,C₁-C₆alkylsulfonyl-C₁-C₆alkyl, aryl, aralkyl, heterocycloalkyl,heteroaryl, or heteroaralkyl;

R₂ and R₄ are C₁-C₆alkoxy;

R₆ is —C(O)R₈, —C(S)R₈, —C(O)OR₈, —C(O)NR₈R₉, —C(S)NR₈R₉, —C(NH)R₈, or—S(O₂)R₈;

R₇ is alkyl, aralkyl, or heteroaralkyl;

R₈ and R₉ each represent independently hydrogen, C₁-C₆alkyl,heteroalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

n represents independently for each occurrence 0, 1, or 2.

In certain embodiments, Z is —CH₂—. In certain embodiments, R₁, R₃, andR₅ each represent independently for each occurrence hydrogen, halogen,hydroxyl, amino, or C₁-C₆alkyl. In certain embodiments, R₂ and R₄ aremethoxy. In certain embodiments, R₆ is C(O)R₈, and R₈ is C₁-C₆alkyl. Incertain embodiments, R₇ is alkyl. In certain embodiments, R₈ and R₉ eachrepresent independently hydrogen or C₁-C₆alkyl. In certain embodiments,n is 0.

In certain embodiments, the Nec-3 related compound is

or a pharmaceutically acceptable salt thereof.

In certain other embodiments, the Nec-3 related compound is

or a pharmaceutically acceptable salt thereof.

The Nec-3 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as in Degterev etal., (2008) NAT CHEM BIOL 4:313-321; and International PatentApplication Publication No. WO 2007/075772, both of which is herebyincorporated by reference.

In certain embodiments, the necrostatin is a Nec-4 related compound ofFormula IV:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

R₁ is

R₂ and R₃ each represent independently for each occurrence hydrogen ormethyl;

R₄ represents independently for each occurrence halogen, hydrogen,C₁-C₆alkyl, C₂-C₆alkenyl, or C₂-C₄alkynyl;

R₅ is C₁-C₄alkyl;

R₆ is hydrogen, halogen, or —CN;

R₇ is hydrogen or C₁-C₄alkyl;

R₈ is C₁-C₆alkyl, or R₈ taken together with R₉, when present, forms acarbocyclic ring;

R₉ is hydrogen or C₁-C₆alkyl, or R₉ taken together with R₈ forms acarbocyclic ring;

R₁₀ is hydrogen or C₁-C₆alkyl;

A is phenylene or a 5-6 membered heteroarylene;

X is N or —C(R₉)—;

Y is N or —C(R₁₀)—;

Z is S or O; and

m and n each represent independently 1, 2, or 3.

In certain embodiments, R₁ is

In certain other embodiments, R₁ is

In certain embodiments, R₂ is hydrogen. In certain embodiments, R₃ ismethyl. In certain other embodiments, R₃ is hydrogen. In certainembodiments, R₄ is halogen, such as fluorine or chlorine. In certainembodiments, R₄ is halogen. In certain embodiments, R₅ is methyl orethyl. In certain embodiments, R₆ is CN. In certain embodiments, A isphenylene. In certain embodiments, X is N. In certain embodiments, Y isN. In certain embodiments, Z is S. In certain embodiments, A isphenylene. In certain embodiments, R₁ is C₁-C₆alkyl, such as methyl. Incertain embodiments, m is 1. In certain embodiments, n is 2.

In certain embodiments, the necrostatin is a Nec-4 related compound ofFormula IV-A:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the necrostatin is a Nec-4 related compound ofFormula IV-B:

or a pharmaceutically acceptable salt thereof.

The Nec-4 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as in Teng etal., (2007) BIOORG MED CITEM LETT, 17: 6836-6840; and Teng et al.,(2008) BIOORG MED CHEM LETT, 18: 3219-3223, both of which areincorporated herein by reference.

In certain embodiments, the necrostatin is a Nec-5 related compound ofFormula V:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

A is a saturated or unsaturated 5-6 membered carbocyclic ring;

X is a bond or C₁-C₄alkylene;

R₁ is C₁-C₆alkyl, halogen, hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄,CO₂R₄, or C(O)N(R₄)₂;

R₂ is

R₃ is —C₁-C₆alkylene-CN, —CN, C₁-C₆alkyl, or C₂-C₆alkenyl;

R₄ represents independently for each occurrence hydrogen, C₁-C₆alkyl,aryl, or aralkyl;

R₅ represents independently for each occurrence C₁-C₆alkyl, halogen,hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄, CO₂R₄, or C(O)N(R₄)₂;

B is a 5-6 membered heterocyclic or carbocylic ring; and

n and p each represent independently 0, 1, or 2.

In certain embodiments, X is a bond. In certain embodiments, A is anunsaturated 6-membered carbocyclic ring. In certain embodiments, R₁ isC₁-C₆alkyl, halogen, hydroxyl, or C₁-C₆alkoxyl. In certain embodiments,R₂ is

such as

In certain embodiments, R₃ is —C₁-C₆alkylene-CN, such as —CH₂—CN. Incertain embodiments, R₄ represents independently for each occurrencehydrogen or C₁-C₆alkyl. In certain embodiments, R₅ representsindependently for each occurrence C₁-C₆alkyl, halogen, hydroxyl, orC₁-C₆alkoxyl. In certain embodiments, B is a 5-6 membered heterocyclicring. In certain embodiments, n is 0. In certain embodiments, p is 0.

In certain embodiments, the necrostatin is a Nec-5 related compound ofFormula V-A:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

R₁ is C₁-C₆alkyl, halogen, hydroxyl, C₁-C₆alkoxyl, or —N(R₄)₂;

R₂ is

R₃ is —C₁-C₆alkylene-CN;

R₄ represents independently for each occurrence hydrogen, C₁-C₆alkyl,aryl, or aralkyl;

R₅ represents independently for each occurrence C₁-C₆alkyl, halogen,hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄, CO₂R₄, or C(O)N(R₄)₂;

B is a 5-6 membered heterocyclic or carbocylic ring; and

n and p each represent independently 0, 1, or 2.

In certain embodiments, the Nec-5 compound is

or a pharmaceutically acceptable salt thereof.

The Nec-5 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as in Degterev etal., (2008) NAT CHEM BIOL 4:313-321; and International PatentApplication Publication No. WO 2008/045406, both of which is herebyincorporated by reference.

In certain embodiments, the necrostatin is a Nec-7 related compound ofFormula VII:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

R₁, R₂, and R₃ each represent independently hydrogen or C₁-C₄alkyl;

R₄ is

R₅ and R₆ each represent independently for each occurrence halogen,C₁-C₆alkyl, hydroxyl, C₁-C₆alkoxyl, —N(R₇)₂, —NO₂, —S—C₁-C₆alkyl,—S-aryl, —SO₂—C₁-C₆alkyl, —SO₂— aryl, —C(O)R₇, —CO₂R₇, —C(O)N(R₇)₂,heterocycloalkyl, aryl, or heteroaryl;

R₇ represents independently for each occurrence hydrogen, C₁-C₆alkyl,aryl, or aralkyl; or two occurrences of R₇ attached to the same nitrogenatom are taken together with the nitrogen atom to which they areattached to form a 3-7 membered heterocyclic ring;

A is a 5-6 membered heterocyclic ring; and

p is 0, 1, or 2.

In certain embodiments, R₁ is hydrogen. In certain embodiments, R₂ ishydrogen. In certain embodiments, R₃ is hydrogen. In certainembodiments, R₄ is

In certain embodiments, R₅ is halogen, C₁-C₆alkyl, hydroxyl,C₁-C₆alkoxyl, or —N(R₇)₂. In certain other embodiments, R₅ is halogen,such as fluorine or chlorine. In certain embodiments, p is 0. In certainother embodiments, R₄ is

such as

In certain embodiments, the Nec-7 related compound is

or a pharmaceutically acceptable salt thereof.

The Nec-7 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as in Zheng etal., in BIOORG MED CITEM LETT, 2008, vol. 18, 4932-4935, which isincorporated herein by reference.

In certain embodiments, the necrostatin is a Nec-7 related compound ofFormula VIII:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

each X¹, X², X³, X⁴, X⁵, and X⁶ is selected, independently, from N orCR^(X1);

each Y¹, Y², and Y³ is selected, independently, from O, S, NR^(Y1), orCR^(Y2)R^(Y3);

each Z¹ and Z² is selected, independently, from 0, S, or NR^(Z1);

each R^(Y1) and R^(Z1) is selected, independently, from H, optionallysubstituted C₁₋₆alkyl, optionally substituted C₂₋₆alkenyl, optionallysubstituted C₂₋₆alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(5A), —C(═O)OR^(5A), or—C(═O)NR^(5A)R^(6A);

-   -   each R^(X1), R^(Y2), and R^(Y3) is selected, independently, from        H, halogen, CN, NC, NO₂, N₃, OR³, SR³, NR³R⁴, —C(═O)R^(5A),        —C(═O)OR^(5A), —C(═O)NR^(5A)R^(6A), —S(═O)R^(5A), —S(═O)₂R^(5A),        —S(═O)₂OR^(5A), —S(═O)₂NR^(5A)R^(6A), optionally substituted        C₁₋₆alkyl, optionally substituted C₂₋₆alkenyl, optionally        substituted C₂₋₆alkynyl, optionally substituted cycloalkyl,        optionally substituted heterocyclyl, optionally substituted        aryl, or optionally substituted heteroaryl;

each R¹, R² R^(5A), R^(5B), R^(6A), and R^(6B) is selected from H,optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆alkenyl,optionally substituted C₂₋₆alkynyl, optionally substituted cycloalkyl,optionally substituted heterocyclyl, optionally substituted aryl, oroptionally substituted heteroaryl; or R^(5A) and R^(6A), or R^(5B) andR^(6B) combine to form a heterocyclyl; and

each R³ and R⁴ is selected from H, optionally substituted C₁₋₆ alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,—C(═O)R^(5B), —C(═S)R^(5B), —C(═NR^(6B))R^(5B), —C(═O)OR^(5B),—C(═O)NR^(5B)R^(6B), —S(═O)R^(5B), —S(═O)₂R^(5B), —S(═O)₂OR^(5B), or—S(═O)²NR^(5B)R^(6B). In certain embodiments, when R¹ is H, X¹, X², andX⁴ are each CH, X³, X⁵, and X⁶ are each N, Y¹ and Y³ are each S, Y² isNH, Z¹ is NH, and Z² is O, then R² is not 4-fluorophenyl.

In certain embodiments, the necrostatin is a Nec-7 related compound ofFormula VIII-A:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

X¹, X², X⁴, X⁵, R¹, Y², and R^(Z1) are as defined for Formula (VIII);

each R^(2A), R^(2B), R^(2C), R^(2D), and R^(2E) is selected,independently, from H, halogen, optionally substituted C₁₋₆ alkyl,optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆alkynyl, optionally substituted cycloalkyl, optionally substitutedheterocyclyl, optionally substituted aryl, optionally substitutedheteroaryl, CN, NC, NO₂, N₃, OR⁷, SR⁷, S(═O)R¹², S(═O)₂R¹², S(═O)OR¹²,S(═O)₂OR¹², NR⁷R⁸, C(═O)R¹², C(═O)OR¹², C(═O)NR¹²R¹³, C(═S)R¹²,C(═S)OR¹², C(═S)NR¹²R¹³, C(═NR⁹)R¹², C(═NR⁹)OR¹², or C(═NR⁹)NR¹²R¹³, orR^(2A) and R^(2B), R^(2B) and R^(2C), R^(2C) and R^(2D), or R^(2D) andR^(2E) combine to form an optionally substituted cycloalkyl or anoptionally substituted heterocyclyl;

each R⁷, R⁸, and R⁹ is selected, independently, from H, optionallysubstituted C₁₄ alkyl, optionally substituted cycloalkyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, S(═O)R¹⁰, S(═O)₂R¹⁰, C(═O)R¹⁰, C(═O)OR¹⁰,C(═O)NR¹⁰R¹¹, C(═S)R¹⁰, C(═S)OR¹⁰, C(═S)NR¹⁰R¹¹, C(═NR¹⁴)R¹⁰,C(═NR¹⁴)OR¹⁰, or C(═NR¹⁴)NR¹⁰R¹¹, or R⁷ and R⁸ combine to form anoptionally substituted heterocyclyl; and

each R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is selected, independently, from H,optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆alkenyl,optionally substituted C₂₋₆alkynyl, optionally substituted cycloalkyl,optionally substituted heterocyclyl, optionally substituted aryl, oroptionally substituted heteroaryl, or R¹⁰ and R¹¹ or R¹² and R¹³ combineto form an optionally substituted heterocyclyl.

In certain embodiments, each R^(2A), R^(2B), R^(2C), R^(2D), and R^(2E)is selected, independently, from H, halogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

In certain embodiments, the necrostatin is a Nec-7 related compoundselected from:

and pharmaceutically acceptable salts thereof.

The Nec-7 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as InternationalPatent Application Publication No. WO 2010/075290, which is herebyincorporated by reference.

In certain embodiments, the necrostatin is a Nec-4 related compound ofFormula IX:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

X₁ and X₂ are, independently, N or CR⁴;

X₃ is selected from O, S, NR⁵, or —(CR⁵)₂;

Y is selected from C(O) or CH₂; and

-   -   is (CR⁶R⁷)_(n);

R₁ is selected from H, halogen, optionally substituted C₁₋₆alkyl, oroptionally substituted C₁₋₆cycloalkyl, or optionally substituted aryl;

R² is selected from H or optionally substituted C₁₋₆alkyl;

R³ is optionally substituted aryl;

each R⁴ is selected from H, halogen, carboxamido, nitro, cyano,optionally substituted C₁₋₆alkyl, or optionally substituted aryl;

R⁵ is selected from H, halogen, optionally substituted C₁₋₆alkyl, oroptionally substituted aryl;

each R⁶ and R⁷ is, independently, selected from H, optionallysubstituted C₁₋₆alkyl, or aryl; and

n is 0, 1, 2, or 3. In certain embodiments, when X₁ and X₂ are N, X₃ isS, Y is C(O), Z is CH₂, R² is H, and R³ is 2-chloro-6-fluoro-phenyl,then R¹ is not methyl.

In certain embodiments, the necrostatin is a Nec-4 related compound ofFormula IX-A:

or a pharmaceutically acceptable salt, ester, or prodrug thereof,wherein:

R¹, R², R³, R⁶ and R⁷ are as defined in Formula (IX).

In certain embodiments, the necrostatin is a Nec-4 related compoundselected from:

and pharmaceutically acceptable salts thereof.

The Nec-4 related compounds described above can be prepared based onsynthetic procedures described in the literature, such as U.S. PatentApplication Publication No. 2009/0099242, which is hereby incorporatedby reference.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 10 or fewer carbonatoms in its backbone (e.g., C₁-C₁₀ for straight chain, C₃-C₁₀ forbranched chain), and alternatively, 5, 4, 3, 2 or 1 carbon atoms in itsbackbone. Likewise, cycloalkyls have from about 3 to about 10 carbonatoms in their ring structure, and alternatively about 5, 6 or 7 carbonsin the ring structure. Exemplary alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,cyclopropyl, and cyclobutyl.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, or —O-alkynyl. The term“alkylene” refers to a diradical of an alkyl group. An exemplaryalkylene group is —CH₂CH₂—.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup.

The term “heteroaralkyl” refers to an alkyl group substituted with aheteroaryl group.

The term “alkenyl” refers to an unsaturated straight or branchedhydrocarbon having at least one carbon-carbon double bond, such as astraight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referredto herein as C₂-C₂alkenyl, C₂-C₁₀alkenyl, and C₂-C₆alkenyl,respectively. Exemplary alkenyl groups include, but are not limited to,vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl,hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl,4-(2-methyl-3-butene)-pentenyl, etc.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-12, 2-8, or 2-6 carbon atoms,referred to herein as C₂-C₁₂alkynyl, C₂-C₈alkynyl, and C₂-C₆alkynyl,respectively. Exemplary alkynyl groups include, but are not limited to,ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl,4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl, etc.

The term “aryl” is art-recognized and refers to a carbocyclic aromaticgroup. Representative aryl groups include phenyl, naphthyl, anthracenyl,and the like. Unless specified otherwise, the aromatic ring may besubstituted at one or more ring positions with, for example, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl,—CO₂alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido,sulfonamide, ketone, aldehyde, ester, heterocyclyl, heteroaryl, —CF₃,—CN, or the like. The term “aryl” also includes polycyclic ring systemshaving two or more carbocyclic rings in which two or more carbons arecommon to two adjoining rings (the rings are “fused rings”) wherein atleast one of the rings is aromatic, e.g., the other cyclic rings may becycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls.

In certain embodiments, the aromatic group is not substituted, i.e., itis unsubstituted.

The term “phenylene” refers to a multivalent radical (e.g., a divalentor trivalent radical) of benzene. To illustrate, a divalent valentradical of benzene is illustrated by the formula

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to saturated, partially unsaturated, or aromatic 3- to 10-memberedring structures, alternatively 3- to 7-membered rings, whose ringstructures include one to four heteroatoms, such as nitrogen, oxygen,and sulfur. Heterocycles may also be mono-, bi-, or other multi-cyclicring systems. A heterocycle may be fused to one or more aryl, partiallyunsaturated, or saturated rings. Heterocyclyl groups include, forexample, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl,dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl,imidazolidinyl, isoquinolyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl,piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuryl,tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl,thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl,lactones, lactams such as azetidinones and pyrrolidinones, sultams,sultones, and the like. Unless specified otherwise, the heterocyclicring is optionally substituted at one or more positions withsubstituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido,amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy,cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl.In certain embodiments, the heterocyclcyl group is not substituted,i.e., it is unsubstituted.

The term “heteroaryl” is art-recognized and refers to aromatic groupsthat include at least one ring heteroatom. In certain instances, aheteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representativeexamples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl,imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl,pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Unless specifiedotherwise, the heteroaryl ring may be substituted at one or more ringpositions with, for example, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl, carbonyl, carboxyl,alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester,heterocyclyl, aryl, —CF₃, —CN, or the like. The term “heteroaryl” alsoincludes polycyclic ring systems having two or more rings in which twoor more carbons are common to two adjoining rings (the rings are “fusedrings”) wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls,and/or aryls.

The term “heteroarylene” refers to a multi-valent (e.g., di-valent ortrivalent) aromatic group that comprises at least one ring heteroatom.An exemplary “heteroarylene” is pyridinylene, which is a multi-valentradical of pyridine. For example, a divalent radical of pyridine isillustrated by the formula

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formula:

wherein R⁵⁰ and R⁵¹ each independently represent hydrogen, alkyl,alkenyl, or —(CH₂)_(m)—R⁶¹; or R⁵⁰ and R⁵¹, taken together with the Natom to which they are attached complete a heterocycle having from 4 to8 atoms in the ring structure; wherein R⁶¹ is aryl, cycloalkyl,cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In certain embodiments, R⁵⁰ and R⁵¹ eachindependently represent hydrogen or alkyl.

The term “amide” or “amido” as used herein refers to a radical of theform —R_(a)C(O)N(R_(b))—, —R_(a)C(O)N(R_(b))R_(c)—, —C(O)NR_(b)R_(c), or—C(O)NH₂, wherein R_(a), R_(b) and R_(c) are each independently selectedfrom alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl,heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, and nitro. Theamide can be attached to another group through the carbon, the nitrogen,R_(b), R_(c), or R_(a). The amide also may be cyclic, for example R_(b)and R_(c), R_(a) and R_(b), or R_(a) and R_(c) may be joined to form a3- to 12-membered ring, such as a 3- to 10-membered ring or a 5- to6-membered ring. The term “carboxamido” refers to the structure—C(O)NR_(b)R_(c).

The term “sulfonamide” or “sulfonamido” as used herein refers to aradical having the structure —N(R_(r))—S(O)₂—R_(s)— or—S(O)₂—N(R_(r))R_(s), where R_(r), and R_(s) can be, for example,hydrogen, alkyl, aryl, cycloalkyl, and heterocyclyl. Exemplarysulfonamides include alkylsulfonamides (e.g., where R_(s) is alkyl),arylsulfonamides (e.g., where R_(s) is aryl), cycloalkyl sulfonamides(e.g., where R_(s) is cycloalkyl), and heterocyclyl sulfonamides (e.g.,where R_(s) is heterocyclyl), etc.

The term “sulfonyl” as used herein refers to a radical having thestructure R_(u)SO₂—, where R_(u) can be alkyl, aryl, cycloalkyl, andheterocyclyl, e.g., alkylsulfonyl. The term “alkylsulfonyl” as usedherein refers to an alkyl group attached to a sulfonyl group.

The symbol “

” indicates a point of attachment.

Unless specified otherwise, the term “optionally substituted” as usedherein means that the specified group may be substituted at one, two ormore positions with, for example, halogen, azide, alkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl,carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide,ketone, aldehyde, ester, heterocyclyl, heteroaryl, —CF₃, —CN, or thelike.

As used herein, the term “therapeutically effective amount” isunderstood to mean the amount of an active ingredient, for example, anecrostatin (e.g., necrostatin-1 or necrostatin-4) and/or a pan-caspaseinhibitor (e.g., Z-VAD or IDN-6556) that is sufficient to reduce,minimize or eliminate the death of retinal ganglion cells associatedwith certain ocular conditions described herein. The compounds of theinvention are administered in amounts effective at, e.g., reducing thedeath of retinal ganglion cells, increasing efficacy compared tomonotherapy with either drug alone, preserving or improving vision,preserving or improving visual function, preventing vision loss, and/orpromoting axon regeneration. It is understood that preserving vision orvisual function, includes stabilizing vision or visual function and/orslowing the decline of vision or visual function prior to treatment.

As used herein, “pharmaceutically acceptable” or “pharmacologicallyacceptable” mean molecular entities and compositions that do not producean adverse, allergic or other untoward reaction when administered to ananimal, or to a human, as appropriate. The term, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

Disclosed herein is a method of preserving the visual function of an eyeof a subject with ocular conditions, wherein a symptom of the ocularcondition is the loss of retinal ganglion cell viability in the retinaof the eye with the conditions. The method comprises (a) administeringto the eye of the subject an effective amount of a necrosis inhibitor(e.g., a necrostatin, e.g., necrostatin-1 or necrostatin-4) and aneffective amount of an apoptosis inhibitor thereby preserving theviability of the retinal ganglion cells disposed within the retina ofthe eye, and (b) measuring the visual function (e.g., visual acuity) ofthe eye after the administration of the necrosis inhibitor and theapoptosis inhibitor. After administration of the necrosis inhibitor andthe apoptosis inhibitor the visual function of the eye may be preservedor improved relative to the visual function of the eye prior toadministration of the necrosis inhibitor and the apoptosis inhibitor.Further, after the administration of the necrosis inhibitor and theapoptosis inhibitor, the preserved retinal ganglion cell is capable ofsupporting axonal regeneration.

Further disclosed is a method of preserving the visual function of aneye of a subject with ocular condition, wherein a symptom of the ocularcondition is the loss of retinal ganglion cell viability in the retinaof the eye with the condition. The method comprises reducing theproduction and/or activity of a RIP-1 kinase and/or a RIP-3 kinase inthe eye to preserve the viability of the retinal ganglion cells disposedwithin the eye. The reduction in the production and/or activity of theRIP-1 kinase and/or RIP-3 kinase may be achieved by administering aneffective amount of a necrosis inhibitor (e.g., RIPK inhibitor, e.g., anecrostatin). The reduction in the production and/or activity of theRIP-1 kinase and/or RIP-3 kinase may be direct (e.g., the necrosisinhibitor modulates the production and/or activity the RIP-1 kinaseand/or RIP-3 kinase directly) or indirect (e.g., the necrosis inhibitoracts upstream of the RIP-1 kinase and/or RIP-3 kinase but theadministration of which indirectly modulates the production and/oractivity of the RIP-1 kinase and/or RIP-3 kinase). Visual function ofthe eye may be measured before and/or after the administration of thenecrosis inhibitor that directly or indirectly reduces the productionand/or activity of a RIP-1 kinase and/or RIP-3 kinase. Afteradministration of the necrosis inhibitor the visual function of the eyemay be preserved or improved relative to the visual function of the eyeprior to administration of the necrosis inhibitor.

In each of the foregoing methods, the ocular condition, wherein asymptom of the condition is the loss of retinal ganglion cell viabilityin the retina of the eye, includes but is not limited to glaucoma, opticnerve injury, optic neuritis, optic neuropathies, diabetic retinopathy,central retinal artery occlusion, and central retinal vein occulusion.It is contemplated that the forgoing methods may be used for thetreatment of optic neuropathies such as ischemic optic neuropathy (e.g.,arteritic or non-arteritic anterior ischemic neuropathy and posteriorischemic optic neuropathy), compressive optic neuropathy, infiltrativeoptic neuropathy, traumatic optic neuropathy, mitochondrial opticneuropathy (e.g., Leber's optic neuropathy), nutritional opticneuropathy, toxic optic neuropathy, and hereditary optic neuropathy(e.g., Leber's optic neuropathy, Dominant Optic Atrophy, Behr'ssyndrome).

Also disclosed is a method of preserving the visual function of an eyeof a subject with an ocular condition selected from the group consistingof glaucoma, optic nerve injury, optic neuropathies, diabeticretinopathy, central retinal artery occlusion, and central retinal veinocculusion. The method comprises administering to the eye of the subjectan effective amount of a necrostatin and an effective amount of anapoptosis inhibitor thereby preserving the viability of the retinalganglion cells disposed within the retina of the eye and the visualfunction of the eye.

In another aspect, disclosed herein is a method of preserving theviability of retinal ganglion cells disposed within a retina of amammalian eye affected by, for example, glaucoma, optic nerve injury,optic neuritis, optic neuropathies, diabetic retinopathy, centralretinal artery occlusion, and central retinal vein occulusion. Themethod comprises administering a necrosis inhibitor and/or an apoptosisinhibitor to the eye in which a region of the retina has been affectedin amounts sufficient to preserve the viability of retinal ganglioncells disposed within the region of the affected retina. The preservedretinal ganglion cell is capable of supporting axonal regeneration.

Also disclosed is a method for promoting axon regeneration in a eye of asubject with an ocular condition, wherein a symptom of the ocularcondition is the loss of retinal ganglion cell viability in the retinaof the eye with the condition. The method comprises administering to theeye of the subject an effective amount of a necrostatin and an effectiveamount of an apoptosis inhibitor thereby promoting axon regeneration ofthe retinal ganglion cell within the retina of the eye.

In each of the foregoing embodiments, it is understood that the methodsand compositions described herein can be used to preserve the viabilityand/or promote axon regeneration of retinal ganglion cells duringtreatment of the underlying conditions including, but not limited to,glaucoma, optic nerve injury, optic neuritis, optic neuropathies,diabetic retinopathy, central retinal artery occlusion, and centralretinal vein occulusion.

Unless specified, the necrostatin can be administered to give a finalconcentration of greater than about 5 μM, for example, in the range ofabout 5 μM to about 1000 μM. In certain embodiments, the necrostatin canbe administered in an amount sufficient to give a final concentration ofnecrostatin in the eye of greater than about 10 μM. In anotherembodiment, the necrostatin can be administered in an amount sufficientto give a final concentration of necrostatin in the eye of greater thanabout 50 μM. In another embodiment, the necrostatin can be administeredin an amount sufficient to give a final concentration of necrostatin inthe eye of greater than about 100 μM. For example, the necrostatin maybe administered in an amount sufficient to give a final concentration ofnecrostatin in the eye in the range from about 5 μM to about 1000 μM, 10μM to about 1000 μM, 50 μM to about 1000 μM, 80 μM to about 1000 μM,about 100 μM to about 1000 μM, about 150 μM to about 1000 μM, from about200 μM to about 800 μM, or from about 200 μM to about 600 μM. In certainembodiments, the necrostatin is administered in an amount sufficient togive a final concentration of necrostatin in the eye of about 400 μM.

The apoptosis inhibitor, for example, the pan-caspase inhibitor, can beadministered in an amount sufficient to give a final concentration ofthe inhibitor in the eye of greater than about 3 μM, for example, in therange of about 3 μM to about 500 μM. In certain embodiments, thenecrostatin can be administered in an amount sufficient to give a finalconcentration of necrostatin in the eye of greater than about 3 μM. Inanother embodiment, the necrostatin can be administered in an amountsufficient to give a final concentration of necrostatin in the eye ofgreater than about 30 μM. In a further embodiment, the necrostatin canbe administered in an amount sufficient to give a final concentration ofnecrostatin in the eye of greater than about 50 μM. In yet a furtherembodiment, the necrostatin can be administered in an amount sufficientto give a final concentration of necrostatin in the eye of greater thanabout 100 μM. For example, the apoptosis inhibitor can be administeredin an amount sufficient to give a final concentration of the inhibitorin the eye in the range from about 3 μM to about 500 μM, from about 80μM to about 500 μM, 100 μM to about 500 μM, 125 μM to about 500 μM, 150μM to about 500 μM or from about 200 μM to about 400 μM. In certainembodiments, apoptosis inhibitor (e.g., the pan-caspase inhibitor) isadministered in an amount sufficient to give a final concentration ofthe inhibitor in the eye of about 300 μM.

In view of the fact that the volume of the eye in a given subject isknown (for example, typical human eye contains 4 to 6 mL of fluid(humor), it is possible to calculate the dosage of the necrostatinand/or the pan-caspase inhibitor to be administered to give thetherapeutically effective concentrations noted above. For example, fromabout 0.035 mg to about 2 mg of necrostatin-1 and from about 0.05 mg toabout 1.5 mg of a pan-caspase inhibitor can be administered to achievethe concentrations noted above.

In certain embodiments, from about 0.025 mg to about 4 mg, from about0.035 mg to about 2 mg, from about 0.05 mg to about 2 mg, from about 0.1mg to about 2 mg, from about 0.2 mg to about 1 mg, or from about 0.2 mgto about 0.8 mg of the necrosis inhibitor (e.g., a necrostatin) can beadministered locally to the eye of a mammal. In one embodiment, 0.5 mgof necrostatin is administered locally to the eye of a mammal. Incertain other embodiments, from about 0.05 mg to about 2 mg, from about0.2 mg to about 2 mg, from about 0.05 mg to about 1.5 mg, from about0.15 mg to about 1.5 mg, from about 0.4 mg to about 1 mg, or from about0.5 mg to about 0.8 mg of an apoptosis inhibitor (e.g., a pan-caspaseinhibitor, e.g., Z-VAD) can be administered locally to the eye of amammal. In certain embodiments, about 0.7 mg of a pan-caspase inhibitor,e.g., Z-VAD, is administered locally to the eye of a mammal.

It is understood that the invention relates to the use of a necrosisinhibitor, either alone or in combination with an apoptosis inhibitor,for preserving the viability and/or promoting the axon regeneration ofretinal ganglion cells for the treatment of the ocular disorder. Theinvention also relates to the use of an apoptois inhibitor, either aloneor in combination with a necrosis inhibitor, for preserving theviability and/or promoting the axon regeneration of retinal ganglioncells for the treatment of the ocular disorder.

In certain aspects, one or more of a necrosis inhibitor, one or more ofan apoptosis inhibitor, or one or more of a necrosis inhibitor and oneor more of an apoptosis inhibitor can be administered to the eye inwhich a region of the retina has been affected in amounts sufficient topreserve the viability and/or promote axon regeneration of retinalganglion cells disposed within the region of the affected retina.

In certain embodiments, the necrosis inhibitor is a necrostatin, forexample, necrostatin-1, a necrostatin-2, a necrostatin-4, anecrostatin-5, and a necrostatin-7. One or more of these necrosisinhibitors can be administered with one or more of the apoptosisinhibitors (e.g., IDN-6556) listed below. Furthermore, it iscontemplated that one or more of the necrostatins shown by Formula I,I-A, I-B, I-C, I-D, I-E, I-F, I-G, II, II-A, III, IV, IV-A, IV-B, V,V-A, VII, VIII, VIII-A, IX, or IX-A can be administered with one or moreof the apoptosis inhibitors (e.g., IDN-6556 or IDN-6734) listed below.

In certain embodiment, the necrosis inhibitor reduces the productionand/or activity of a RIP-1 kinase and/or a RIP-3 kinase. RIP kinaseinhibitors (e.g., RIP-1 kinase and/or RIP-3 kinase inhibitors) asdisclosed herein may further include RNAs, including small inhibitoryRNAs (siRNAs) and short hairpin RNAs (shRNAs). Methods for designing andsynthesizing siRNAs and shRNAs are well known in the art. ExemplaryRIP-1 kinase inhibitors include, for example, a pSIREN-RIP-1 shRNAconstruct which targets RIP-1 kinase as disclosed in Kaiser et al.,(2008) JOURNAL OF IMMUNOLOGY 181:6427-6434. Exemplary RIP-3 kinaseinhibitors include, for example, sc-61482-SH and sc-135170 availablefrom Santa Cruz Biotechnology. In another example, RIP kinase inhibitors(e.g., RIP-1 kinase and/or RIP-3 kinase inhibitors) as disclosed hereinmay include inhibitor of apoptosis proteins (IAPs), active fragmentsthereof, and nucleic acids encoding the same. It is well establishedthat IAPs inhibit RIP-1 kinase by functioning as a E3 ligase for RIP-1kinase (see, for example, Vanlangenakker et al., (2010)).

In certain embodiments, the one or more apoptosis inhibitors may includea pan-caspase inhibitor. The pan-caspase inhibitor can be Z-VAD (i.e.,Z-Val-Ala-Asp(OMe)-CH₂F*), IDN-6556 available from ConatusPharmaceuticals (i.e.,(3-{2-[(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid)(3-{2-[(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid), IDN-6734 available from Conatus Pharmaceuticals, VX-799 availablefrom Vertex Pharmaceuticals, MX1013 and MX2060 derivatives availablefrom Maxim Pharmaceuticals, M-920 available from Merck-Frosst,small-molecule compounds available from Gemin X Pharmaceuticals, RGDpeptides from Merck-Frost and Maxim Pharmaceuticals, or any other knownpan-caspase inhibitor.

Alternatively, the pan-caspase inhibitor can be a cocktail of caspaseinhibitors including two or more specific caspase inhibitors (e.g.,synthetic caspase inhibitors) such as a caspase 1 inhibitor, a caspase 2inhibitor, a caspase 3 inhibitor, a caspase 4 inhibitor, a caspase 5inhibitor, a caspase 6 inhibitor, a caspase 7 inhibitor, a caspase 8inhibitor, and a caspase 9 inhibitor. It is contemplated that one ormore of the pan-caspase inhibitors may be used in combination with oneor more necrostatins (e.g., necrostain-1 and/or necrostatin-4).

Exemplary synthetic caspase 1 inhibitors, include, for example,Ac-N-Me-Tyr-Val-Ala-Asp-aldehyde (SEQ ID NO: 7),Ac-Trp-Glu-His-Asp-aldehyde (SEQ ID NO: 8),Ac-Tyr-N-Me-Val-Ala-N-Me-Asp-aldehyde (SEQ ID NO: 9),Ac-Tyr-Val-Ala-Asp-Aldehyde (SEQ ID NO: 10),Ac-Tyr-Val-Ala-Asp-chloromethylketone (SEQ ID NO: 11),Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone (SEQ ID NO: 12),Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetol (SEQ ID NO: 13),Ac-Tyr-Val-Lys-Asp-aldehyde (SEQ ID NO: 14),Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone (SEQ IDNO: 15), biotinyl-Tyr-Val-Ala-Asp-chloromethylketone (SEQ ID NO: 16),Boc-Asp(OBzl)-chloromethylketone,ethoxycarbonyl-Ala-Tyr-Val-Ala-Asp-aldehyde (pseudo acid) (SEQ ID NO:17), Z-Asp-2,6-dichlorobenzoyloxymethylketone,Z-Asp(OlBu)-bromomethylketone, Z-Tyr-Val-Ala-Asp-chloromethylketone (SEQID NO: 18), Z-Tyr-Val-Ala-DL-Asp-fluoromethlyketone (SEQ ID NO: 19),Z-Val-Ala-DL-Asp-fluoromethylketone, andZ-Val-Ala-DL-Asp(OMe)-fluoromethylketone, all of which can be obtainedfrom Bachem Bioscience Inc., PA. Other exemplary caspase 1 inhibitorsinclude, for example, Z-Val-Ala-Asp-fluoromethylketone,biotin-X-Val-Ala-Asp-fluoromethylketone, Ac-Val-Ala-Asp-aldehyde,Boc-Asp-fluoromethylketone,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Pro-Tyr-Val-Ala-Asp-aldehyde(SEQ ID NO: 1), biotin-Tyr-Val-Ala-Asp-fluoroacyloxymethylketone (SEQ IDNO: 20), Ac-Tyr-Val-Ala-Asp-acyloxymethylketone (SEQ ID NO: 21),Z-Asp-CH2-DCB, and Z-Tyr-Val-Ala-Asp-fluoromethylketone (SEQ ID NO: 22),all of which are available from Calbiochem, IDN-11104 available fromConatus Pharmaceuticals, and VX-740 and VX-756 available from VertexPharmaceuticals.

Exemplary synthetic caspase 2 inhibitors, include, for example,Ac-Val-Asp-Val-Ala-Asp-aldehyde (SEQ ID NO: 23), which can be obtainedfrom Bachem Bioscience Inc., PA, andZ-Val-Asp-Val-Ala-Asp-fluoromethylketone (SEQ ID NO: 24), which can beobtained from Calbiochem, Calif.

Exemplary synthetic caspase 3 precursor protease inhibitors include, forexample, Ac-Glu-Ser-Met-Asp-aldehyde (pseudo acid) (SEQ ID NO: 25) andAc-Ile-Glu-Thr-Asp-aldehyde (pseudo acid) (SEQ ID NO: 26) which can beobtained from Bachem Bioscience Inc., PA. Exemplary synthetic caspase 3inhibitors include, for example, Ac-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO:27), Ac-Asp-Met-Gln-Asp-aldehyde (SEQ ID NO: 28),biotinyl-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO: 29),Z-Asp-Glu-Val-Asp-chloromethylketone (SEQ ID NO: 30),Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone (SEQ ID NO: 31),and Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone which can be obtained fromBachem Bioscience Inc., PA. Other exemplary caspase 3 inhibitorsinclude, for example,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde(SEQ ID NO: 2), biotin-X-Asp-Glu-Val-Asp-fluoromethylketone (SEQ ID NO:32), Ac-Asp-Glu-Val-Asp-chloromethylketone (SEQ ID NO: 33), all of whichare available from Calbiochem. Another exemplary caspase 3 inhibitorincludes, the caspase 3 inhibitorN-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyketone(z-Asp-Glu-Val-Asp-fmk) (SEQ ID NO: 34), which is available from EnzymeSystems Products. Additional exemplary caspase 3 inhibitors includeM-826 and M-791 available from Merck-Frosst, Immunocasp-3, Ad-G/iCasp3,and PEF-F8-CP3.

Exemplary synthetic caspase 4 inhibitors include, for example,Ac-Leu-Glu-Val-Asp-aldehyde (SEQ ID NO: 35) andZ-Tyr-Val-Ala-DL-Asp-fluoromethylketone (SEQ ID NO: 36), which can beobtained from Bachem Bioscience Inc., PA, andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-Val-Pro-aldehyde(SEQ ID NO: 3), which can be obtained from Calbiochem, Calif.

Exemplary synthetic caspase 5 inhibitors include, for example,Z-Trp-His-Glu-Asp-fluoromethylketone (SEQ ID NO: 37), which can beobtained from Calbiochem, Calif., and Ac-Trp-Glu-His-Asp-aldehyde (SEQID NO: 38) and Z-Trp-Glu(O-Me)-His-Asp(O-Me) fluoromethylketone (SEQ IDNO: 39), which can be obtained from Sigma Aldrich, Germany.

Exemplary synthetic caspase 6 inhibitors include, for example,Ac-Val-Glu-Ile-Asp-aldehyde (SEQ ID NO: 40),Z-Val-Glu-Ile-Asp-fluoromethylketone (SEQ ID NO: 41), andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Glu-Ile-Asp-aldehyde (SEQ ID NO: 4),which can be obtained from Calbiochem. Another exemplary caspase 6inhibitor includes Immunocasp-6.

Exemplary synthetic caspase 7 inhibitors include, for example,Z-Asp(OMe)-Gln-Met-Asp(OMe) fluoromethylketone (SEQ ID NO: 42),Ac-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO: 43),Biotin-Asp-Glu-Val-Asp-fluoromethylketone (SEQ ID NO: 44),Z-Asp-Glu-Val-Asp-fluoromethylketone (SEQ ID NO: 45),Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde(SEQ ID NO: 2), which can be obtained from Sigma Aldrich, Germany.

Exemplary synthetic caspase 8 inhibitors include, for example,Ac-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO: 46), Ac-Ile-Glu-Pro-Asp-aldehyde(SEQ ID NO: 47), Ac-Ile-Glu-Thr-Asp-aldehyde (SEQ ID NO: 48),Ac-Trp-Glu-His-Asp-aldehyde (SEQ ID NO: 49) andBoc-Ala-Glu-Val-Asp-aldehyde (SEQ ID NO: 50) which can be obtained fromBachem Bioscience Inc., PA. Other exemplary caspase 8 inhibitorsinclude, for example,Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Glu-Thr-Asp-aldehyde(SEQ ID NO: 5) and Z-Ile-Glu-Thr-Asp-fluoromethylketone (SEQ ID NO: 51),which can be obtained from Calbiochem, Calif.

Exemplary synthetic caspase 9 inhibitors, include, for example,Ac-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO: 52), Ac-Leu-Glu-His-Asp-aldehyde(SEQ ID NO: 53), and Ac-Leu-Glu-His-Asp-chloromethylketone (SEQ ID NO:54) which can be obtained from Bachem Bioscience Inc., PA. Otherexemplary caspase 9 inhibitors include, for example,Z-Leu-Glu-His-Asp-fluoromethylketone (SEQ ID NO: 55) andAc-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-His-Asp-aldehyde(SEQ ID NO:6), which can be obtained from Calbiochem, Calif. Anotherexemplary caspase 9 inhibitor includes FKBP12/caspase-9 fusion protein.

The pan-caspase inhibitor may also be an endogenous caspase inhibitor ora combination of an endogenous caspase inhibitor with one or moresynthetic caspase inhibitors. For example, one useful class ofendogenous caspase inhibitor includes proteins known as inhibitors ofapoptosis proteins (IAPs) (Deveraux et al., (1998) EMBO J. 17(8):2215-2223) including bioactive fragments and analogs thereof. Oneexemplary IAP includes X-linked inhibitor of apoptosis protein (XIAP),which has been shown to be a direct and selective inhibitor ofcaspase-3, caspase-7 and caspase-9. Another exemplary IAP includessurvivin (see, U.S. Pat. No. 6,245,523; Papapetropoulos et al., (2000)J. BIOL. CHEM. 275: 9102-9105), including bioactive fragments andanalogs thereof. Survivin has been reported to inhibit caspase-3 andcaspase-7 activity.

In certain embodiments, the one or more apoptosis inhibitors may targetthe inhibitor of apoptosis proteins (IAPs) and secondmitochondria-derived activator of caspases (SMACs). Exemplary apoptosisinhibitors that target IAPs and SMACs, include, for example, BIR3antagonists available from Idun Pharmaceuticals, capped tripeptide XIAPantagonists from Abbot Laboratories, TWX024, polyphenylurea derivatives,SMAC-mimetic compounds, embelin, XIAP antisense and RNAi constructs,AEG35156/GEM 640 available from Aegera Therapeutics, HIV-Tat- andpolyarginine conjugated SMAC peptides, and nonpeptide small-moleculeSMAC mimetics. It is contemplated that one or more of the apoptosisinhibitors which target IAPs and SMACs may be used in combination withone or more necrostatins (e.g., necrostain-1 and/or necrostatin-4).

In certain embodiments, the one or more apoptosis inhibitors may targetthe TNF-related apoptosis-inducing ligand (TRAIL) receptors. Exemplaryapoptosis inhibitors that target the TRAIL receptors, include, forexample, HGS-ETR1, HGS-ETR2, and HGS-TR2J available from Human GenomeSciences, and PRO1762 available from Amgen. It is contemplated that oneor more of the apoptosis inhibitors which target the TRAIL receptors maybe used in combination with one or more necrostatins (e.g., necrostain-1and/or necrostatin-4).

In certain embodiments, the one or more apoptosis inhibitors may targetCD95/Fas. Exemplary apoptosis inhibitors that target CD95/FAS, include,for example, CD95-Fc available from ApoGenix GmbH. It is contemplatedthat one or more of the apoptosis inhibitors which target CD95/Fas maybe used in combination with one or more necrostatins (e.g., necrostain-1and/or necrostatin-4).

In certain embodiments, the one or more apoptosis inhibitors may be ananti-FasL factors. Exemplary anti-FasL factors include, for example,anti-FasL neutralizing antibody (available, for example, fromPharmingen, San Diego, Calif.); peptides and nucleic acids (for example,anti-FasL aptamers) that bind FasL to prevent or reduce its binding toits cognate receptor; certain antibodies and antigen binding fragmentsthereof and peptides that bind preferentially to the Fas receptor;antisense nucleotides and double stranded RNA for RNAi that ultimatelyreduce or eliminate the production of either FasL or the Fas receptor;soluble Fas; soluble FasL; decoy receptor-3 (DcR3) and analoguesthereof; matrix metalloproteinases (MMPs); vasoactive intestinal peptide(VIP); pituitary adenylate cyclase-activating polypeptide (PACAP);forskolin; combined use of benazepril and valsartan; nonpeptidiccorticotropin-releasing hormone receptor type 1 (CRH-R1)-specificantagonists; mimosine; peptides that produce a defective Fas-FasLcomplex; platelet-activating factor (PAF); and endothelin-1 (ET-1).These anti-FasL factors can act as direct or indirect antagonists ofFasL activity.

In certain embodiments, the one or more apoptosis inhibitors may targetthe tumor necrosis factor (TNF). Exemplary apoptosis inhibitors thattarget TNF, include, for example, recombinant TNF-α, adalimumabavailable from Abbott, infliximab available from Centocor Ortho BiotechInc., etanercept from Amgen, CDP571 available from Celltech, and ISIS104838 (a 2′-O-methoxyethyl antisense construct against TNF-alpha)available from ISIS Pharmaceuticals. It is contemplated that one or moreof the apoptosis inhibitors which target TNF may be used in combinationwith one or more necrostatins (e.g., necrostain-1 and/or necrostatin-4).

In certain embodiments, the one or more apoptosis inhibitors may targetsurvivin. Exemplary apoptosis inhibitors that target survivin, include,for example, LY2181308 available from ISIS Pharmaceuticals andAd-survivin T34A. It is contemplated that one or more of the apoptosisinhibitors which target survivin may be used in combination with one ormore necrostatins (e.g., necrostain-1 and/or necrostatin-4).

In certain embodiments, the one or more apoptosis inhibitors may targetthe Bcl-2 proteins. Exemplary apoptosis inhibitors that target the Bcl-2proteins, include, for example, Bcl-2 blockers available from IdunPharmaceuticals and Abbot Laboratories, Gx01 series of compoundsavailable from Gemin X Pharmaceuticals, Bcl-2 small-molecule antagonist,Tetrocarcin-A derivatives available from Kyowa Hakko Kogyo Co.,Chelerythrine, antimycin A derivatives, HA14-1, synthetic compoundbinding to the BH3 of Bcl-2, Genasense available from Sanofi-Aventis,ISIS 22783 available from ISIS Pharmaceuticals, bispecific Bcl-2/Bcl-XLantisense, BH3 peptides from Bax, Bak, Bid or Bad, SAHBs, and BH3Is. Itis contemplated that one or more of the apoptosis inhibitors whichtarget the Bcl-2 proteins may be used in combination with one or morenecrostatins (e.g., necrostain-1 and/or necrostatin-4).

In certain embodiments, the one or more apoptosis inhibitors may targetp53. Exemplary apoptosis inhibitors that target p53, include, forexample, INGN201 available from Invitrogen Therapeutics, SCH58500available from Schering-Plough, ONYX-015 available from OnyxPharmaceuticals, C-terminal p53 peptides, CDB3, Amifostine, CP31398available from Pfizer, Prima-1, HPF E6-binding peptide aptamers, Nutlinsavailable from Roche, Chalcones, Small peptide compounds, andPifithrin-α. It is contemplated that one or more of the apoptosisinhibitors which target p53 may be used in combination with one or morenecrostatins (e.g., necrostain-1 and/or necrostatin-4).

In certain embodiments, it is contemplated that one or more necrostatins(e.g., necrostatin-1 and/or necrostatin-4) may be used in combinationwith a pan-caspase inhibitor. For example, in one embodiment,necrostain-1 and/or necrostatin-4 may be used in combination with Z-VADavailable from R&D Systems (Cat. No. FMK001) and Promega (Cat. No.G7231). In another embodiment, necrostain-1 and/or necrostatin-4 may beused in combination with IDN-6556 available from ConatusPharmaceuticals. In yet another embodiment, necrostain-1 and/ornecrostatin-4 may be used in combination with IDN-6734 available fromConatus Pharmaceuticals.

In certain embodiments, it is contemplated that one or more necrostatins(e.g., necrostatin-1 and/or necrostatin-4) may be used in combinationwith a TNF inhibitor. For example, in one embodiment, necrostain-1and/or necrostatin-4 may be used in combination with adalimumabavailable from Abbot Laboratories. In another embodiment, necrostain-1and/or necrostatin-4 may be used in combination with etanerceptavailable from Amgen, Inc. In yet another embodiment, necrostain-1and/or necrostatin-4 may be used in combination with infiximab availablefrom Centocor Ortho Biotech, Inc.

In certain embodiments, it is contemplated that one or more necrostatins(e.g., necrostatin-1 and/or necrostatin-4) may be used in combinationwith a p53 agonist. For example, in one embodiment, necrostain-1 and/ornecrostatin-4 may be used in combination with INGN 201 available fromInvitrogen Therapeutics. In another embodiment, necrostain-1 and/ornecrostatin-4 may be used in combination with nutlins, for example,nutlin-3 available from Cayman Chemical (Cat. No. 10004372). In yetanother embodiment, necrostain-1 and/or necrostatin-4 may be used incombination with CP31398 available from Tocris Bioscience (Cat. No.3023).

In certain embodiments, it is contemplated that one or more necrostatins(e.g., necrostatin-1 and/or necrostatin-4) may be used in combinationwith an anti-FasL factor. For example, in one embodiment, necrostain-1and/or necrostatin-4 may be used in combination with anti-FasLneutralizing antibody available from Pharmingen (San Diego, Calif.).

As shown in FIG. 1, depending upon the specific apoptotic inhibitorchosen, it is possible that the apoptotic inhibitor can modulate boththe apoptotic and necrotic pathways, and depending upon the specificnecrosis inhibitor chosen, it is possible that the necrosis inhibitorcan modulate both the necrotic and apoptotic pathways. For example, aRIP-1 inhibitor may inhibit both necrotic and apoptotic cell death thuspreserving the viability of the retinal ganglion cells in the retina ofthe eye of a subject with an ocular condition as disclosed herein.

As discussed herein, the methods and compositions of the invention canpreserve the visual function of an eye of a subject with an ocularcondition. Assessment of axonal regeneration may also be monitoredthrough visual function tests as disclosed herein. Visual function canbe measured using one or more of a variety of methods well-known in theart. For example, visual function can be assessed by measuring visualacuity. Visual acuity can be assessed, for example, by usingconventional “eye charts” in which visual acuity is evaluated by theability to discern letters of a certain size, with five letters of agiven size present on each line (see, e.g., the “ETDRS” eye chartdescribed in the Murphy, R. P., CURRENT TECHNIQUES IN OPTHALMIC LASERSURGERY, 3^(rd) Ed., edited by L. D. Singerman, and G. Cascas,Butterworth Heinemann, 2000). Evaluation of visual acuity may also beachieved by measuring reading speed and reading time. Visual acuity maybe measured to evaluate whether administration of a necrosis inhibitorand/or an apoptosis inhibitor to the affected eye preserves or permitsimprovement of visual acuity (e.g., to 20/40 vision or to 20/20 vision).

Visual function may also be measured by determining whether there is anincrease in the thickness of the Nerve Fiber layer (NFL) (e.g., NFLthickness is 15% thicker than, 35% thicker than, 50% thicker than, 60%thicker than, 70% thicker than, or 80% thicker than a macula without thetreatment as measured by optical coherence tomography (OCT); animprovement of the ganglion cell layer or bipolar cell layer orphotoreceptor cell layer or its subdivisions as seen in the OCT; animprovement of visual field (e.g., by at least 10% in the mean standarddeviation on the Humphrey Visual Field Test; an improvement of anelectroretinograph (ERG), a measurement of the electrical response ofthe retina to light stimulation, (e.g., to increase ERG amplitude by atleast 15%); and/or preservation or improvement of multifocal ERG, whichevaluates the response of the retina to multifocal stimulation andallows characterization of the function of a limited area of the retina.

Visual function may also be measured by electrooculography (EOG), whichis a technique for measuring the resting potential of the retina. EOG isparticularly useful for the assessment of RPE function. EOG may be usedto evaluate whether administration of a necrosis inhibitor and/or anapoptosis inhibitor to the retina of the affected eye preserves orpermits improvement in, for example, the Arden ratio (e.g., an increasein Arden ratio of at least 10%).

Visual function may also be assessed through fundus autofluorescence(AF) imaging, which is a clinical tool that allows evaluation of theinteraction between photoreceptor cells and the RPE. For example,increased fundus AF or decreased fundus AF has been shown to occur inAMD and other ocular disorders. Fundus AF imaging may be used toevaluate whether administration of a necrosis inhibitor and/or anapoptosis inhibitor to the retina of the affected eye slows diseaseprogression.

Visual function may also be assessed by evaluation of contrastsensitivity, which a measurement of the ability to discern betweenluminances of different levels in a static image. An evaluation ofcontrast sensitivity may be used to assess whether administration of anecrosis inhibitor and/or an apoptosis inhibitor to the retina of theaffected eye preserves or permits improvement in the resolving power ofthe eye.

Visual function may also be assessed by microperimetry, which monitorsretinal visual function against retinal thickness or structure and thecondition of the subject's fixation over time. Microperimetry may beused to assess whether administration of a necrosis inhibitor and/or anapoptosis inhibitor to the retina of the affected eye preserves orpermits improvement in retinal sensitivity and fixation.

In certain embodiments, the necrosis inhibitor, e.g., a necrostatin,and/or the apoptosis inhibitor, e.g., a pan-caspase inhibitor such asZ-VAD or IDN-6556, may be administered locally to the eye of a subject,e.g., a human subject, following other treatments of the retina topreserve or to permit improvement of visual acuity (e.g., to 20/40vision or to 20/20 vision); to increase the thickness of the macula(e.g., macula thickness is 15% thicker than, 35% thicker than, 50%thicker than, 60% thicker than, 70% thicker than, or 80% thicker than amacula without the treatment as measured by optical coherence tomography(OCT)); to permit improvement in the appearance and structure of thephotoreceptor cell layer and its supporting RPE, to permit theimprovement of visual field (e.g., by at least 10% in the mean standarddeviation on the Humphrey Visual Field Test; and/or to permit theimprovement of an electroretinograph (ERG), a measurement of theelectrical response of the retina to light stimulation, (e.g., toincrease ERG amplitude by at least 15%).

In certain embodiments, the necrosis inhibitor, e.g., a necrostatin,and/or the apoptosis inhibitor, e.g., a pan-caspase inhibitor such asZ-VAD or IDN-6556, are administered locally to the eye of a mammal byintravitreal injection. Both agents may be administered on the day ofdiagnosis and/or the same day that the retina has gone through othertreatments and/or in the post-operative period. The agents may then beadministered every three days, every five days, or every seven daysuntil the mammal, e.g., a human, has improved vision (e.g., visualacuity has improved to 20/40 vision or to 20/20 vision), the thicknessof the NFL or macula has increased (e.g., macula thickness is 15%thicker than, 35% thicker than, 50% thicker than, 60% thicker than, 70%thicker than, or 80% thicker than without treatment as measured by OCT);the appearance of the ganglion cell layer or bipolar cell layer orphotoreceptor cell layer and RPE as detected by OCT; the visual fieldhas improved by at least 10% in the mean standard deviation asdetermined by Humphrey Visual Field testing; and/or the mammal's retinashows an increased response to light stimulation (e.g., at least a 15%increase in amplitude as determined by electroretinography).

The necrosis inhibitor and the apoptosis inhibitor can be administeredby the same route or by different routes. The necrosis inhibitor and/orthe apoptosis inhibitor may be administered locally to the eye, forexample, by intravitreal, intraocular, intraorbital, subconjuctival,subretinal or transscleral routes. For example, the necrosis inhibitorand/or the apoptosis inhibitor may be administered locally to the eye byintravitreal injection. It is contemplated that local modes ofadministration may reduce or eliminate the incidence of potential sideeffects (e.g., systemic toxicity) that may occur during systemicadministration.

Alternatively, the necrosis inhibitor and/or the apoptosis inhibitor maybe administered systemically, e.g., by oral or parenteral routes.Parenteral routes include, for example, intravenous, intrarterial,intramuscular, intradermal, subcutaneous, intranasal and intraperitonealroutes.

The necrosis inhibitor and the apoptosis inhibitor may be administeredto a subject simultaneously or sequentially. It will be appreciated thatwhen administered simultaneously, the necrosis inhibitor and theapoptosis inhibitor may be in the same pharmaceutically acceptablecarrier (e.g., solubilized in the same viscoelastic carrier that isintroduced into the eye) or the two drugs may be dissolved or dispersedin separate pharmaceutical carriers, which are administered at the sametime. Alternatively, the drugs may be provided in separate dosage formsand administered sequentially. For example, in some embodiments, thenecrostatin may be administered before the pan-caspase inhibitor. Inother examples, the pan-caspase inhibitor may be administered before thenecrostatin. In addition, it is appreciated that, in some embodiments, asingle active agent may inhibit both necrosis and apoptosis.

Administration may be provided as a periodic bolus (for example,intravitreally or intravenously) or as continuous infusion from aninternal reservoir (for example, from an implant disposed at an intra-or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242))or from an external reservoir (for example, from an intravenous bag, ora contact lens slow release formulation system). The necrosis inhibitorand/or the apoptosis inhibitor may be administered locally, for example,by continuous release from a sustained release drug delivery deviceimmobilized to an inner wall of the eye or via targeted transscleralcontrolled release into the choroid (see, for example, PCT/US00/00207,PCT/US02/14279, Ambati et al., (2000) INVEST. OPHTHALMOL. VIS. SCI.41:1181-1185, and Ambati et al., (2000) INVEST. OPHTHALMOL. VIS. SCI.41:1186-1191). A variety of devices suitable for administering thedisclosed necrosis and/or apoptosis inhibitors locally to the inside ofthe eye are known in the art. See, for example, U.S. Pat. Nos.6,251,090, 6,299,895, 6,416,777, 6,413,540, and 6,375,972, andPCT/US00/28187.

The necrosis inhibitor and/or the apoptosis inhibitor may be solubilizedin a carrier, for example, a viscoelastic carrier, that is introducedlocally into the eye. One or both inhibitors also may be administered ina pharmaceutically acceptable carrier or vehicle so that administrationdoes not otherwise adversely affect the recipient's electrolyte and/orvolume balance. The carrier may comprise, for example, physiologicsaline or other buffer system. In exemplary embodiments, thenecrostatin, the pan-caspase inhibitor, or both the necrostatin and thepan-caspase inhibitor may be solubilized in PBS or another aqueousbuffer by sonication. Alternatively, one or both drugs may besolubilized using conventional solvent or solubilization systems, forexample, dimethyl sulfoxide (DMSO), dimethoxyethane (DME),dimethylformamide (DMF), cyclodextran, micelles, liposomes, liposomalagents, and other solvents known in the art to aid in the solubilizationand administration of hydrophobic agents.

In other embodiments, the necrosis inhibitor and/or the apoptosisinhibitor may be solubilized in a liposome or microsphere. Methods fordelivery of a drug or combination of drugs in liposomes and/ormicrospheres are well-known in the art.

In addition, it is contemplated that the necrosis inhibitor and/or theapoptosis inhibitor may be formulated so as to permit release of one orboth inhibitors over a prolonged period of time. A release system caninclude a matrix of a biodegradable material or a material, whichreleases the incorporated active agents. The active agents can behomogeneously or heterogeneously distributed within a release system. Avariety of release systems may be useful in the practice of theinvention, however, the choice of the appropriate system will dependupon the rate of release required by a particular drug regime. Bothnon-degradable and degradable release systems can be used. Suitablerelease systems include polymers and polymeric matrices, non-polymericmatrices, or inorganic and organic excipients and diluents such as, butnot limited to, calcium carbonate and sugar (for example, trehalose).Release systems may be natural or synthetic. However, under certaincircumstances, synthetic release systems are preferred because generallythey are more reliable, more reproducible and produce more definedrelease profiles. The release system material can be selected so thatinhibitors having different molecular weights are released by diffusionthrough or degradation of the material.

Representative synthetic, biodegradable polymers include, for example:polyamides such as poly(amino acids) and poly(peptides); polyesters suchas poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolicacid), and poly(caprolactone); poly(anhydrides); polyorthoesters;polycarbonates; and chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), copolymers and mixtures thereof.Representative synthetic, non-degradable polymers include, for example:polyethers such as poly(ethylene oxide), poly(ethylene glycol), andpoly(tetramethylene oxide); vinyl polymers-polyacrylates andpolymethacrylates such as methyl, ethyl, other alkyl, hydroxyethylmethacrylate, acrylic and methacrylic acids, and others such aspoly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate);poly(urethanes); cellulose and its derivatives such as alkyl,hydroxyalkyl, ethers, esters, nitrocellulose, and various celluloseacetates; polysiloxanes; and any chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), copolymers and mixtures thereof.

One of the primary vehicles currently being developed for the deliveryof ocular pharmacological agents is the poly(lactide-co-glycolide)microsphere for intraocular injection. The microspheres are composed ofa polymer of lactic acid and glycolic acid, which are structured to formhollow spheres. These spheres can be approximately 15-30 μm in diameterand can be loaded with a variety of compounds varying in size fromsimple molecules to high molecular weight proteins such as antibodies.The biocompatibility of these microspheres is well established (see,Sintzel et al., (1996)), and microspheres have been used to deliver awide variety of pharmacological agents in numerous biological systems.After injection, poly(lactide-co-glycolide) microspheres are hydrolyzedby the surrounding tissues, which cause the release of the contents ofthe microspheres (Zhu et al., (2000)). As will be appreciated, the invivo half-life of a microsphere can be adjusted depending on thespecific needs of the system.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present invention also consistessentially of, or consist of, the recited components, and that theprocesses of the present invention also consist essentially of, orconsist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions are immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

EXAMPLES

The invention is further illustrated by the following examples, whichare provided for illustrative purposes only, and should not be construedas limiting the scope or content of the invention in any way.

In the examples described herein, all animal experiments adhered to theAssociation for Research in Vision and Ophthalmology Statement for theUse of Animals in Ophthalmic and Vision Research, and protocols wereapproved by the Animal Care Committee of the Massachusetts Eye and EarInfirmary. Wild-type C57BL/6 mice were purchased from Charles RiverLaboratories (Wilmington, Mass.). The mice were fed standard laboratorychow and allowed free access to water in an air-conditioned room with a12 hour light/12 hour dark cycle. RIP-3 knockout (RIP-3−/−) mice werekindly provided by Dr. V. M. Dixit (Genentech, San Francisco, Calif.).RIP-3−/− mice were generated as described previously and backcrossed toC57BL/6 mice (Newton et al., (2004) MOL CELL BIOL 24:1464-1469). Exceptas noted otherwise, the animals were anesthetized with ketaminehydrochloride (30 mg/kg; Ketalar, Parke-Davis, Morris Plains, N.J.) andxylazine hydrochloride (5 mg/kg; Rompun, Harver-Lockhart, Morris Plains,N.J.) before all experimental manipulations.

Two mouse models of RGC death were utilized: optic nerve (ON) injury andN-methyl-D-aspartate (NMDA) retinal excitotoxicity. ON injury leads toRGC death as a result of primary damage to the axons, with its ensuingdisruption of retrograde axonal transport (Libby et al., (2005) PLOSGENET 1:17-26) and is used as an RGC death model. In the ON injurymodel, mice were anesthetized and subjected to severe crush injury at1-2 mm distance from the eyeball for 15 seconds using cross-actionforceps taking special care not to interfere with the blood supply(Levkovitch-Verbin, (2004) EYE 18:1066-1074). Injured mice were randomlydivided into 4 groups for treatment: vehicle group (0.5% DMSO and 0.8%cyclodextrin in PBS, n=6), ZVAD group (300 n=6), Nec-1 group (4 mM,n=6), and ZVAD plus Nec-1 group (n=6). Soon after injury, each groupreceived an intravitreal injection (2 μl) with the respective compounds.In the NMDA retinal excitotoxicity model, NMDA (2 μl of a 0.1M stock;Sigma-Aldrich) was injected intravitreously in combination with ZVAD,Nec-1, or ZVAD plus Nec-1 and mice were separated into theaforementioned 4 groups. The dose of these compounds was selected basedon previous studies (Karl et al., (2008) PROC NATL ACAD SCI USA105:19508-19513; Knoferle et al., (2010) PROC NATL ACAD SCI USA107:6064-6069; Nakazawa et al., (2006) J NEUROSCI 26:12633-12641;Rosenbaum et al., (2010) J NEUROSCI RES 88:1569-1576).

The following reagents were utilized: Z-VAD (Alexis, Plymouth MeetingPa.), IDN-6556 (kindly provided by TetraLogics Pharmaceuticals), a Nec-1compound of Formula I-C (a kind gift from Dr. J. Yuan, Harvard MedicalSchool, Boston, Mass.), and a Nec-4 compound of Formula IV-A (kindlyprovided by TetraLogics Pharmaceuticals).

ON-injured mice received an intravitreal injection of 3-methyladenine(3-MA), in DMSO (33.3 mM; Sigma-Aldrich, St. Louis, Mo.), a goatanti-mouse TNF-α blocking antibody, or the appropriate control goatantibody (R & D Systems, Minneapolis, Minn.).

Intravitreal injections were performed as follows. Briefly, the tip of a33 gauge needle (Hamilton, Reno, Nev.) was carefully inserted throughthe sclera into the intravitreal space to reduce intraocular pressure.Then, the needle was extracted, loaded with compounds and tangentiallyreinserted through the sclera into the intravitreal space, inducing aself-sealing wound tunnel. After injection, the absence of choroidalbleeding was confirmed. At specified times after injury, mice weresacrificed with an overdose of sodium pentobarbital, and eyes wereenucleated.

Total RNA extraction, cDNA synthesis and PCR amplification have beenperformed as previously reported (Kayama et al., (2010) OPHTHALMIC RES43:79-91). A real-time PCR assay was performed with Prism 7700 SequenceDetection System (Applied Biosystems, Foster City, Calif.). The primersare shown below in Table 1.

TABLE 1 II. Taqman Gene Expression Assays Protein Assay ID # SupplierRIP1 Mm00436354_m1 Applied Biosystems RIP3 Mm00444947_m1 AppliedBiosystems TNF-α Mm99999068_m1 Applied Biosystems Atg5 Mm00504340_m1Applied Biosystems Atg7 Mm00512209_m1 Applied Biosystems Atg12Mm00503201_m1 Applied Biosystems

For relative comparison of each gene, the Ct value of real-time PCR datawas analyzed with the ΔΔCt method and normalized to an endogenouscontrol ((3-actin).

For Western blot analysis, whole retinas were harvested and lysed for 30min on ice in lysis buffer (50 mM Tris-HCl [pH 8], with 120 mM NaCl and1% Nonidet P-40), supplemented with a mixture of proteinase inhibitors(Complete Mini; Roche Diagnostics, Basel, Switzerland). Thirtymicrograms of protein per sample were separated in a 4-20% gradientsodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (InvitrogenCorporation, Carlsbad, Calif., USA) electrophoresis and the proteinswere electroblotted onto PVDF membranes. After 20 min incubation inblocking solution (Starting Block™ T20, Thermo Scientific, Waltham,Mass.), membranes were incubated with primary antibodies overnight at 4°C. Peroxidase-labeled secondary antibodies (Amersham Pharmacia Biotech,Piscataway, N.J., USA) were used and proteins were visualized withenhanced chemiluminescence technique (Amersham Pharmacia Biotech). Fulllist of primary antibodies and working concentrations are shown below inTable 2.

TABLE 2 I. Antibodies Immuno- Immuno- Antigen Host blot stainingSupplier RIP3 Rabbit 1:10000 No Sigma-Aldrich RIP1 Mouse 1:1000 No BDBiosciences LC3 Rabbit 1:1000 No Cell Signalling LC3 Goat No 1:50 SCBTBm3b Goat No 1:50 SCBT Caspase-9 Rabbit No 1:300 Cell SignallingCaspase-3 Rabbit No 1:300 Cell Signalling TNF-α Goat No No R&D (blockingantibody) β-tubulin Rabbit 1:1000 No Cell Signalling

TUNEL and quantification of TUNEL (+) cells were performed as previouslydescribed (Nakazawa et al., (2007) PROC NATL ACAD SCI USA 104:2425-2430)by using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110;Chemicon International, Temecula, Calif.).

All values disclosed were expressed as the mean±SD. Statisticaldifferences between two groups were analyzed by Mann-Whitney U test.Multiple group comparison was performed by ANOVA followed byTukey-Kramer adjustments. Differences were considered significant atP<0.05.

Example 1: Efficacy of a Necrosis Inhibitor and a Pan-Caspase Inhibitorin the Treatment of Glaucoma and Optic Nerve Injury

Glaucoma is a group of ocular disorders, characterized by optic nerveinjury. In most cases of glaucoma, the optic nerve injury is caused byelevated intraocular pressure. Furthermore, higher intraocular pressuresare generally associated with greater nerve damage. In this example,mouse models of optic nerve (ON) injury was used to assess the role ofRIP-mediated programmed necrosis and apoptosis in this ocular disorder.Given that ON injury is a hallmark of glaucoma, this model also providedan assessment of RIP-mediated programmed necrosis in RGC loss inglaucoma.

A. Neutralization of TNF-α Prevents Optic Nerve Injury-Induced RGC Death

Recent evidence has demonstrated the role of TNF-α as a mediator ofretinal ganglion cell (RGC) death in glaucoma (Nakazawa et al., (2006) JNEUROSCI 26:12633-12641)). Furthermore, in the glaucomatous eye, deathreceptors of TNF-α are up-regulated in RGCs and optic nerve axons (Yanet al., (2000), Yuan et al., (2000), Tezel et al., (2001) INVESTOPHTHALMOL VIS SCI 42:1787-1794)). To investigate whether TNF-α isinvolved in RGC cell death in mice subjected to optic nerve (ON) injury(induced by physical injury to the optic nerve), TNF-α level in theretina was measured using quantitative real-time RT-PCR. As depicted inFIG. 2A, at one day after ON injury, TNF-α mRNA levels increased almost10- to 13-fold relative to non-injured mice.

An anti-TNF-α neutralizing antibody (0.1 mg/ml) or a control antibodywas injected into the vitreous of mice that underwent ON injury. Oneweek following injection, the number of RGCs were measured. In addition,inner plexiform layer (IPL) thickness (a location of RGC axons) was alsoassessed. Specifically, eyes were enucleated and RGC loss was quantifiedfrom histological sections of mouse retina. Only transverse sectionsinvolving the optic disc were used for analysis and the fieldscorresponding to approximately 400 μm of both sides of retina extendingfrom the ON head (2 points/section×3 sections per eye, n=6) wereexamined with an optical microscope (×40 objectives). IPL thickness wasmeasured with OpenLab software (Open Lab, Florence, Italy) (2points/section×3 sections per eye, n=6). The ratio of IPL thickness wascalculated as a percentage of IPL thickness in the normal mouse eyes(n=6).

Mice treated with the control antibody showed significant reduction bothin RGCs and IPL thickness (FIGS. 2B-2D), whereas, mice treated withTNF-α neutralizing antibody showed negligible loss of RGCs (FIGS. 2B and2C) and minimal change in IPL thickness (FIGS. 2B and 2D). These dataindicate that TNF-α plays a critical role in RGC death after ON injury.

B. ON Injury Induces Changes in RIP3 and RIP1 Expression

TNF-α has been shown to be a potent inducer of programmed necrosis aswell as apoptosis (Degterev et al., (2005) NAT CHEM BIOL 1:112-119,Balkwill, (2009)). The kinases RIP3 and RIP1 are key signaling moleculesin cellular apoptotic and necrotic pathways and are regulated by TNF-α(He et al., (2009) CELL 137:1100-1111, Vandenabeele et al., (2010) SCISIGNAL 3:re4)). Thus, RIP3 and RIP1 mRNA levels were measured in theretina of mice with ON injury using quantitative RT-PCR. One day afterON injury, expression of RIP3 and RIP1 increased significantly up to 9-and 5-fold, respectively, compared to non-injured mice (FIGS. 2E and2F). RIP3 and RIP1 protein levels were also assessed using Western Blotanalysis. After ON injury, expressions of RIP3 and RIP1 were found to beapproximately 3-fold up-regulated compared with non-injured retina(FIGS. 2G-2I). These results suggest that RIP kinases may contribute toON injury-induced RGC death.

C. Simultaneous Inhibition of RIP Kinases and Caspases is Required forPreventing RGC Death

In human glaucoma, RGC death has been attributed mainly to apoptosis(Kerrigan et al., (1997), Tatton et al., (2001)). Indeed, activation ofcaspase-dependent apoptotic pathways has been demonstrated to occurafter ON injury (Kermer et al., (2000)). The activities of caspase-8,caspase-9 and caspase-3 were measured using a commercially available kitaccording to the manufacturer's instructions (APT171/131/139; Millipore,Billerica, Mass.). As seen in FIG. 3R, the activities of caspase-3,caspase-8, and caspase-9 were significantly increased one day after ONinjury when compared with non-injured retina. In line with a previousreport (Chauvier et al., (2007)), broad caspase inhibition with Z-VAD(benzoyl-Val-Ala-Asp-fluoromethyl ketone) failed to rescue RGCs one dayafter ON injury (FIGS. 3A and 3B), despite its ability to decrease theactivities of the caspases by 70% (FIGS. 3H-3K).

Nec-1, which is a potent and selective inhibitor of programmed necrosistargeting RIP1 kinase activity (Degterev et al., (2008)), was employedto investigate the effect of RIP kinase inhibition in RGC death after ONinjury. Mice received an intravitreal injection of Z-VAD (300 uM) and/orNec-1 (400 uM). The dose of these compounds was selected based onstudies that established that their half-life inside the eye is around 6hours. Administration of Nec-1 and/or Z-VAD did not affect increase ofTNF-α levels after ON injury (FIG. 3L).

By day one after ON injury, there was a dramatic decrease in apoptoticRGCs in mice that received a combination of Z-VAD and Nec-1 (FIGS. 3Aand 3B). Injection of Nec-1 alone, did not affect the number of TUNELpositive cells (FIGS. 3A and 3B), which declined significantly at daythree and day seven after ON injury (FIGS. 3M and 3N).

Immunohistochemistry was performed to assess the number of remainingviable RGCs using an antibody against the specific RGC marker Brn3b.Although TUNEL positive cells peaked at day one after injury,significant decrease in RGC number was first observed on day three(FIGS. 3O and 3P). One week after ON injury, the number of Brn3bpositive cells in the vehicle group was decreased by approximately 50%compared to the group that received a combination of Z-VAD with Nec-1(FIGS. 3C and 3D).

Inner plexiform layer (IPL) thickness and ganglion cell layer (GCL) withIPL thickness (location of RGC cell bodies and axons) were measuredusing immunohistochemistry and a Spectral Domain Optical CoherenceTomography (SD-OCT) system (Bioptigen Inc., Durham, N.C.). Mice werepositioned on a custom cassette, which allowed three-dimensional freerotation and alignment of the mouse eye for imaging. Hydration withnormal saline was used to preserve corneal clarity. A volume analysiscentered on the ON head was performed, using 100 horizontal, raster, andconsecutive B-scan lines, each one composed by 1200 A-scans. Thethickness of GCL+IPL was assessed at 500 μm, 400 μm, 300 μm distancefrom the ON head (nasally and temporally) as well as at 200 μm, 400 μmabove and below the ON head. Co-administration of Z-VAD and Nec-1remarkably reversed the decrease in IPL thickness (FIG. 3Q) and GCL withIPL thickness that was seen in the vehicle group (FIGS. 3E-3G). Takentogether, these data suggest that inhibition of RIP kinases togetherwith a broad-spectrum caspase inhibitor are required for effectiveneuroprotection after ON injury.

D. Broad Caspase Inhibition Shifts Cell Death from Apoptosis to Necrosis

To investigate further the underlying mechanism of why Z-VAD fails toprevent RGC death after ON injury, propidium iodide (PI) was used todetect necrotic cells in GCL (Unal Cevik et al., (2010)). At one dayafter ON injury, Z-VAD administration increased the number of PIpositive cells compared with vehicle (FIGS. 4A and 4B). In comparison,the number of PI positive cells was significantly decreased after Nec-1co-administration (FIGS. 4A and 4B). These results provide directevidence that Z-VAD treatment shifts RGC death from apoptotic tonecrotic death.

In addition, the morphology of RGC death was assessed by transmissionelectron microscopy (TEM) as previously described (Trichonas et al.,(2010) PROC NATL ACAD SCI USA)). Specifically, the eyes were enucleated,and the posterior segments were fixed in 2.5% glutaraldehyde and 2%paraformaldehyde in 0.1 M cacodylate buffer with 0.08 M CaCl₂ at 4° C.The eyes were post-fixed for 1.5 hours in 2% aqueous OsO4, dehydrated inethanol and water, and embedded in EPON. Ultrathin sections were cutfrom blocks and stained with saturated, aqueous uranyl acetate andSato's lead stain. The specimens were observed with a Philips CM10electron microscope. More than 50 RGCs per eye were photographed andsubjected to quantification of cell death in a masked fashion. RGCsshowing cellular shrinkage and nuclear condensation were defined asapoptotic cells, and RGCs associated with cellular and organelleswelling and discontinuities in nuclear and plasma membrane were definedas necrotic cells. Electron dense granular materials were labeled simplyas end-stage unclassified cell death. Autophagosome was defined as adouble- or multi-membraned structure containing cytoplasmic materialand/or organelles, and autolysosome was defined as cytoplasmic vesiclecontaining electron dense degraded material, as previously described(Eskelinen, 2008).

RGC death was categorized into apoptosis, necrosis and unclassifiedend-stage of death, as previously described (Trichonas et al., (2010)PROC NATL ACAD SCI USA). Consistent with the PI study, at one day afterON injury, both apoptotic and necrotic RGC death was observed in thevehicle-treated retina (apoptotic cells: 13.4±5.8%, necrotic cells:16.9±4.2%, unclassified: 2.2±2.4%; FIGS. 3A and 3B). Nec-1 treatmentslightly decreased necrotic RGC death (% apoptotic cells: 13.0±8.4%,necrotic cells: 10.6±2.6%, unclassified: 1.0±1.8%; FIGS. 4C and 4D). Incontrast, ZVAD treatment significantly decreased apoptotic RGC death,while it increased necrotic cell death without reducing overall cellloss (% apoptotic cells: 5.6±3.4%, necrotic cells: 30.7±4.9%,unclassified: 3.4±2.3, P<0.01; FIGS. 4C and 4D). Further, infiltrationof inflammatory cells was more prevalent in ZVAD-treated retina (FIG.4C). Co-administration of ZVAD and Nec-1 led to a substantial decreaseof both apoptotic and necrotic RGC death (% apoptotic cells: 4.2±5.1%,necrotic cells: 9.8±6.6%, unclassified: 1.8±1.8%, P<0.01; FIGS. 4C and4D).

These results demonstrate that RIP1 kinase mediated necrosis is animportant pathway of RGC death in addition to apoptosis and compensatesfor blockage of caspase-dependent apoptosis after ON injury.

E. RIP3 Deficiency Attenuates RGC Loss after ON Injury

To further elucidate the role of RIP kinase pathway in RGC death afterON injury, RIP3 deficient mice were used (He et al., (2009) CELL137:1100-1111, Zhang et al., (2009) SCIENCE 325:332-336)), becauseRIP1^(−/−) mice die postnatally at day 1-3 (Kelliher et al., (1998)IMMUNITY 8:297-303)). At one day after ON injury, RIP3^(−/−) miceexhibited significantly less TUNEL positive cells 1 day compared towildtype controls (FIGS. 5A and 5B). Z-VAD administration in RIP3^(−/−)mice lead to a marked decrease in TUNEL positive cells compared to wildtype and RIP3^(−/−) mice that received vehicle solution. Moreover, Nec-1co-administration in RIP3^(−/−) mice did not have any additional effect,whereas administration by itself did not have any effect at all (FIGS.5A and 5B).

The number of viable RGCs were assessed by Brn3b immunohistochemistryand measurement of IPL thickness. Seven days after ON injury, RIP3^(−/−)mice (vehicle, black box) demonstrated increased number of Brn3bpositive cells compared to wildtype mice, (vehicle, white box) which wasfurther enhanced with Z-VAD administration (FIGS. 5C and 5D). At oneweek after ON injury, RIP3 deficiency combined with Z-VAD administrationalso resulted in preservation of IPL thickness compared to RIP3^(−/−)mice that received only vehicle solution (FIGS. 5C and 5E). The numberof Brn3b positive cells and IPL thickness did not alter after Nec-1coadministration (FIGS. 5C-5E).

To investigate whether RIP3 deficiency can prevent the switch fromapoptotic to necrotic RGC death after Z-VAD treatment, PI staining wasperformed. PI staining shows the number of cells with disrupted plasmamembrane. RIP3 deficient mice exhibited significantly less PI positivecells compared with Z-VAD treated WT mice (FIGS. 5F and 5G). This resultsuggests that RIP3 kinase plays an essential role in ON injury-inducedprogrammed necrosis, especially in the presence of caspase inhibitor.

F. Inhibition of Caspase and RIP Kinase Prevents RGC Death after NMDAInjury

Glutamatergic excitotoxicity has been implicated as a mechanism of RGCdeath in glaucoma (Dreyer et al., (1996) ARCH OPHTHALMOL 114:299-305))and intravitreous N-methyl-D-aspartate (NMDA) injection is used as anexcitotoxic RGC insult model (Libby et al., (2005) PLOS GENET 1:17-26)),which is a different model of glaucoma compared to the ON injury modeldiscussed above. To investigate the role of caspases and RIP kinases inanother model of RGC death, we examined the effect of ZVAD plus Nec-1 inthe NMDA injury model. Consistent with results of the ON injury model,TNF-α mRNA levels increased at 1 day after NMDA injury over 10-foldcompared with non-injured mice (FIG. 6A). Quantitative real-time PCR andWestern blot analyses revealed that expressions of RIP3 and RIP1 afterNMDA injury were up-regulated compared with those in non-injured retina(FIGS. 6B-6D). Treatment with ZVAD or Nec-1 alone decreased the numberof TUNEL positive cells and prevented the reduction of Brn3b positivecells, and these protective effects were further enhanced byco-administration of ZVAD plus Nec-1 (FIGS. 6E-6H). Rip3 deficiency alsodecreased RGC loss after NMDA injury (FIGS. 6E-6H). These resultssuggest that caspase and RIP kinase pathways are critical inducers ofRGC death in NMDA injury as well as ON injury.

G. RIP Kinases Mediate Apoptosis-Inducing Factor (AIF) NuclearTranslocation after ON Injury

Apoptosis inducing factor (AIF) is a mitochondrial flavoprotein which isinvolved in initiating caspase-independent apoptosis. After loss ofmitochondrial membrane potential, AIF translocates into the nucleus,induces DNA fragmentation and peripheral chromatin condensation (Susinet al., (1999), Susin et al., (2000), Candé et al., (2004)). To analyzewhether mitochondrial release of AIF is associated with RGC death afterON injury, immunostaining for AIF was performed together with TUNELassay. One day after ON injury, there was a prominent nucleartranslocation of AIF, which was not affected by pan-caspase inhibition(FIGS. 7A and 7B). In contrast, co-administration of Z-VAD with Nec-1 orRIP3 deficiency substantially prevented nuclear translocation of AIF(FIGS. 7A and 7B). These results indicate that AIF is heavily involvedin RGC death after ON injury and RIP kinases play a crucial role in itsnuclear translocation.

It has been reported that RIP kinases control ROS generation duringprogrammed necrosis (Cho et. al., (2009) CELL 137:1112-1123, Zhang etal., (2009) SCIENCE 325:332-336)). To investigate whether ROS play aroll in oxidative retinal damage after ON injury, protein level ofcarbonyl contents was analyzed using ELISA using the OxiSelect™ ProteinCarbonyl ELISA Kit (Cell Biolabs, San Diego, Calif.). One day after ONinjury, carbonyl contents dramatically increased in the treatment ofZ-VAD group compared with vehicle group (FIG. 9C). In contrast, noincrease in carbonyl contents was seen with mice co-administered withZ-VAD and Nec-1 or with RIP3^(−/−) mice (FIG. 9C). These resultsindicated that RIP kinases have a crucial role of AIF translocation intonuclear via ROS production.

H. Ultrastructural Changes of RGCs after ON Injury

It is widely accepted that cell death occurs through the morphologicallydistinct processes of apoptosis, necrosis, or autophagic cell death(Kroemer et al., (2009) CELL DEATH DIFFER 16:3-11)). Apoptosis ischaracterized by activation of caspases, DNA fragmentation, and membraneblebbing (Kroemer et al., (2009) CELL DEATH DIFFER 16:3-11, Yi et al.,(2009)), whereas necrosis is characterized by swelling of theendoplasmic reticulum, mitochondria, and cytoplasm, with subsequentrupture of the plasma membrane and lysis of the cells (Kroemer et al.,(2009) CELL DEATH DIFFER 16:3-11, Festjens et al., (2006)). AlthoughTUNEL has been used traditionally as a marker of apoptosis, a previousreport has shown that necrosis, programmed or otherwise, also yields DNAfragments that react with TUNEL in vivo, rendering it difficult todistinguish between apoptosis and necrosis (Grasl-Kraupp et al., (1995)HEPATOLOGY 21:1465-1468)).

To identify the RGC death mode seen after ON injury transmissionelectron microscopy (TEM) studies was performed as previously described.RGCs that were unable to be classified were defined as end-stage celldeath/unclassified. Under normal conditions, RGCs had a well-definedcontinuous plasma membrane, and a non-uniform distribution of organellesin the cytoplasm, with maximum concentration in the perinuclear region.RGCs contained tubular sacs of rough endoplasmic reticulum (rER)surrounded by large numbers of ribosomes (Nissl bodies). Mitochondriawere identified as round or oval double-membrane structures withcharacteristic cristae. In addition, the cytoplasm contained elements ofGolgi apparatus (GA), free ribosomes and microtubules sectioned atvarious angles. A large round nucleus, surrounded by a double-layerednuclear membrane, contained homogeneously dispersed karyoplasm(chromatin material) and one or two electron dense nucleoli (FIG. 8A)(Saggu et al., (2010) BMC NEUROSCI 11:97)). After ON injury, RGC deaththrough apoptosis was predominant. As can be seen in FIG. 8B,autophagosomes and autolysosomes were observed especially in necroticcells with cellular swelling (FIG. 8B).

Administration of Z-VAD blocked apoptotic cell death and sensitizedcells to necrotic cell death. Nec-1 treatment did not influence theratio of apoptotic to necrotic death. In comparison, co-administrationof Nec-1 with Z-VAD prevented the switch from apoptotic to necrotic celldeath; thus, ameliorating RGC loss.

I. RIP kinases Mediate Autophagic Cell Death After ON Injury

TEM data indicated that autophagic cell death is also involved in RGCdeath after ON injury. Autophagic cell death is recognized by theformation of autophagosomes, double-membrane autophagic vacuoles thateventually fuse with lysosomes to form autolysosomes (Levine et al.,(2004)). In this type of cell death, the Atg6-Vps34 complex wassuggested to be critical for autophagosome-vesicle nucleation (Levine etal., (2005)). Elongation of the autophagosomal membrane formed by theAtg6-Vps34 complex is assisted by two Atg12 and Atg8 ubiquitin-likeconjugation systems (Ichimura et al., (2000)). Atg12, activated by Atg7,covalently attaches to Atg5, forming the irreversible conjugate.

To investigate whether autophagy influences RGC death after ON injury,expression of several genes critical for autophagosome formationincluding Atg5, 7 and 12 was measured by quantitative RT-PCR. Thesetranscripts were found to be up-regulated 2.0- to 2.5-fold at one dayafter ON injury (FIGS. 9A-9C). ZVAD administration further increasedAtg12 expression compared with vehicle treatment (FIG. 9C). In contrast,administration of ZVAD plus Nec-1, or Rip3 deficiency suppressedexpression of Atg5, 7 and 12 transcripts (FIGS. 9A-9C).

Protein levels of LC3, an autophagy marker, were also assessed bywestern blot analysis. In the vehicle, LC3-II, an isoform associatedwith autophagy activity (Kabeya et al, 2000), was up-regulated byapproximately 30% compared with non-injured mice. Consistent withresults from the quantitative PCR analysis, protein level of LC3-IIincreased more than vehicle following Z-VAD administration (FIG. 9D).

Immunohistochemistry was performed to confirm the localization of LC3.In the non-injured retina, expression of LC3 showed faintly detectablestaining (FIG. 9E). However, some LC3 positive cells were detected inthe GCL one day after ON injury (FIG. 9E).

Together, these findings indicate that autophagosome formation increasesin RGCs after ON injury and part of this process may be associated withRIP1 kinase-mediated necrosis. This is consistent with previous reportswhich showed that inhibition of autophagic cell death rescues cell death(Yu et al., (2004) SCIENCE 304:1500-1502, Knoferle et al., (2010) PROCNATL ACAD SCI USA 107:6064-6069)).

J. Inhibitor of Autophagic Cell Death Partially Suppressed RGCs Deathafter ON Injury

To investigate the role of autophagy in RGC death after ON injury, weexamined the effect of 3-methyladenine (3-MA) on RGCs death after ONinjury. 3-MA inhibits autophagosome formation (Seglen and Gordon, (1982)PROC NATL ACAD SCI USA 79:1889-1892)). One day after ON injury, TUNELassay was performed. The number of TUNEL positive cells had a small butstatistically significant decline in mice treated with 3-MA compared tothe non-treated mice. In addition, the number of Brn3b positive cellsand IPL thickness were also analyzed. At seven days after ON injury,3-MA treatment significantly prevented the reduction of Brn3b positivecells and IPL thickness ratio, although this effect was less than thatof the ZVAD plus Nec-1 combination treatment (FIGS. 10C-10E). Toinvestigate whether ROS play a role in oxidative retinal damage after ONinjury, protein level of carbonyl contents was analyzed using an ELISAkit (Cell Biolabs, San Diego, Calif.) according to the manufacturer'sinstructions. One day after ON injury, carbonyl contents decreased inthe 3-MA treated group compared with untreated control (FIG. 10F).Together, these results suggest that RGCs loss was attenuated after ONinjury by inhibition of autophagic cell death.

Example 2: Efficacy of a Necrosis Inhibitor and a Pan-Caspase Inhibitorin Promoting RGC Survival and Axon Regeneration

Like most pathways in the mature central nervous system, the optic nervecannot regenerate if injured, leaving victims of traumatic nerve injuryor degenerative diseases such as glaucoma with life-long visual losses.This situation can be, at least, partially reversed by enhancing theintrinsic growth state of retinal ganglion cells (RGCs). In thisexample, the efficacy of necrosis inhibitor and a pan-caspase inhibitorin promoting RGC survival and axon regeneration is investigated using amouse optic nerve crush model.

A. A Necrosis Inhibitor in Combination with a Caspase Inhibitor PromotesRGC Survival in a Optic Nerve Crush Model

Mice were subjected to optic nerve crush surgery. Specifically, animalswere anesthetized with an intraperitoneal injection of ketamine (60-80mg/kg: Phoenix Pharmaceutical, St. Joseph, Mo.) and xylazine (10-15mg/kg: Bayer, Shawnee Mission, K A). Animals were positioned in astereotaxic apparatus and a 1-1.5 cm incision was made in the skin abovethe right orbit. Under microscopic illumination, the lachrymal glandsand extraocular muscles were resected to expose 3-4 mm of the opticnerve. The epineurium was slit open along the long axis, and the nervewas crushed 2 mm behind the eye with angled jeweler's forceps (Dumont#5) for 10 seconds, avoiding injury to the ophthalmic artery. Nerveinjury was verified by the appearance of a clearing at the crush site,while the vascular integrity of the retina was evaluated by fundoscopicexamination. Cases in which the vascular integrity of the retina was inquestion were excluded from the study.

Following surgery, mice were divided into four groups for treatment:vehicle group, ZVAD group (300 μM; given at day 0, day 3 and day 7 afterinjury), Nec-1 group (4 mM; given at day 0, day 3 and day 7 afterinjury), and ZVAD plus Nec-1 group (300 μM and 4 mM, respectively; giveneither once or at day 0, day 3 and day 7 after injury). Soon afterinjury, each group received an intravitreal injection (3 μl) with therespective compounds. As a control, one group of mice were injected withZymosan (12.5 μg/μl), a yeast cell wall preparation, known to stimulateaxonal regeneration.

Fourteen days following injection, the number of RGCs were measured byBrn3a staining. Specifically, eyes were enucleated and RGC loss wasquantified from histological sections of mouse retina. Images of eightprespecified areas, 2 mm from the optic disc, were captured underfluorescent illumination (2 points/section×4 sections per eye, n=8)using a camera (Nikon E800). Brn3a-positive cells were counted using NIHImageJ software.

As seen in FIG. 11, a combination of ZVAD and Nec-1 significantlyprevented RGC death and promoted RGC survival following optic nervecrush injury when compared to treatment with Zymosan alone (p<0.05). Theeffect of the ZVAD and Nec-1 combination treatment on RGC survival waseven more pronounced when the treatment was given at day 0, day 3 andday 7 after injury when compared to a single treatment at day 0(p<0.05).

B. A Necrosis Inhibitor in Combination with a Caspase Inhibitor PromotesAxon Regeneration

To investigate the efficacy of necrosis inhibitor and pan-caspaseinhibitor in promoting axon regeneration, eight-weeks-old mice weresubjected to optic nerve crush surgery as previously described.Subsequently, injured mice were divided into five groups of treatment:vehicle group, ZVAD group (300 μM; given at day 0, day 3 and day 7 afterinjury), Nec-1 group (4 mM; given at day 0, day 3 and day 7 afterinjury), ZVAD plus Nec-1 group (300 μM and 4 mM, respectively; givenonce at day 0), and ZVAD plus Nec-1 group (300 μM and 4 mM,respectively; given at day 0, day 3 and day 7 after injury).

Axon regeneration was assessed by obtaining longitudinal sections of theoptic nerve and counting the number of axons at pre-specified distancesfrom the injury site. Specifically, mice were sacrificed at 14 daysafter optic nerve injury and were perfused with saline and 4%paraformaldehyde (PFA). Optic nerves and eyes were dissected andpostfixed in PFA. Nerves were impregnated with 10% and then 30% sucrose,embedded in OCT Tissue Tek Medium (Sakura Finetek), frozen, cut in thelongitudinal plane at 14 μm, and mounted on coated slides. Regeneratingaxons were visualized by staining with a sheep antibody to βIII-tubulin,followed by staining with a fluorescently labeled secondary antibody.Axons were counted manually in at least eight longitudinal sections percase at pre-specified distances from the injury site. The number ofregenerating axons at various distances are determined as describedpreviously (Leon et al., (2000) J NEUROSCI 20:4615-4626). To determinethe number of surviving cells, staining with an anti-Brn3a antibody wasused.

FIGS. 12A-12E show longitudinal sections of the optic nerve followingoptic nerve crush injury. The sections are stained with an antibodyagainst PIII-tubulin, which marks axon fibers. In each photograph, anarrow indicates the sites of optic nerve injury, and staining beyond theinjury site starting from left to right indicates axon regeneration(e.g., axons regenerate from the site of injury into the nerve). Nosignificant axon regeneration was seen in mice treated with vehiclecontrol, as demonstrated by the lack of axon staining (FIG. 12A).Treatment with Nec-1 or ZVAD alone had minimal effects on axonregeneration (FIGS. 12B and 12C). In contrast, ZVAD plus Nec-1combination treatment significantly enhanced axon outgrowth asdemonstrated by the increase in axon staining (FIGS. 12D and 12E; seethe regions denoted by the horizontal reference lines under eachfigure). Further, as shown in FIGS. 12D and 12E, the effect of the ZVADand Nec-1 combination treatment on axon regeneration was more pronouncedwhen the treatment was given at day 0, day 3 and day 7 after injury whencompared to a single treatment at day 0. These results indicate thatZVAD and Nec-1 combination treatment not only ameliorates the loss ofRGC following optic nerve injury, but also promotes axon regenerationfollowing injury.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles cited herein are incorporated by reference in their entiretyfor all purposes.

EQUIVALENTS

The invention can be embodied in other specific forms with departingfrom the essential characteristics thereof. The foregoing embodimentstherefore are to be considered illustrative rather than limiting on theinvention described herein. The scope of the invention is indicated bythe appended claims rather than by the foregoing description, and allchanges that come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

We claim:
 1. A method of preserving visual function of an eye of asubject with an ocular condition selected from the group consisting ofan optic neuropathy and diabetic retinopathy, wherein a symptom of theocular condition is the loss of retinal ganglion cell viability in theretina of the eye with the condition, the method comprising: (a)administering to the eye of the subject an effective amount of anecrosis inhibitor selected from the group consisting of necrostatin-1,necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5,necrostatin-7, and related compounds, and an effective amount of anapoptosis inhibitor selected from the group consisting of a pan-caspaseinhibitor, a caspase-1 inhibitor, a caspase-3 inhibitor, a caspase-8inhibitor, and a caspase-9 inhibitor, thereby to preserve the viabilityof a retinal ganglion cell disposed within the retina of the eye; and(b) after step (a), measuring visual function of the eye.
 2. The methodof claim 1, wherein, after administration of the necrosis inhibitor andthe apoptosis inhibitor, the visual function of the eye is preserved orimproved relative to the visual function prior to administration of thenecrosis inhibitor and the apoptosis inhibitor. 3-9. (canceled)
 10. Themethod of claim 2, wherein the visual function is visual acuity. 11-14.(canceled)
 15. The method of claim 1, wherein the ocular condition is anoptic neuropathy selected from the group consisting of an ischemic opticneuropathy, compressive optic neuropathy, infiltrative optic neuropathy,traumatic optic neuropathy, a mitochondrial optic neuropathy,nutritional optic neuropathy, toxic optic neuropathy, and a hereditaryoptic neuropathy.
 16. The method of claim 1, wherein the ocularcondition is diabetic retinopathy. 17-18. (canceled)
 19. The method ofclaim 1, wherein the necrosis inhibitor is necrostatin
 1. 20. The methodof claim 1, wherein the necrosis inhibitor is administered to provide afinal concentration of the necrosis inhibitor in the eye greater thanabout 10 μM. 21-25. (canceled)
 26. The method of claim 1, wherein fromabout 0.05 mg to about 2 mg of the necrosis inhibitor is administered.27-29. (canceled)
 30. The method of claim 1, wherein the pan-caspaseinhibitor is zVAD, IDN-6556, or a combination thereof. 31-36. (canceled)37. The method of claim 1, wherein from about 0.15 mg to about 1.5 mg ofthe pan-caspase inhibitor is administered. 38-39. (canceled)
 40. Themethod of claim 1, wherein the necrosis inhibitor, the apoptosisinhibitor, or both the necrosis inhibitor and the apoptosis inhibitorare administered to the eye.
 41. (canceled)
 42. The method of claim 1,wherein the necrosis inhibitor, the apoptosis inhibitor, or both thenecrosis inhibitor and the apoptosis inhibitor are administered byintraocular injection.
 43. The method of claim 42, wherein the necrosisinhibitor, the apoptosis inhibitor, or both the necrosis inhibitor andthe apoptosis inhibitor are administered intravitreally.
 44. (canceled)45. The method of claim 1, wherein the necrosis inhibitor, the apoptosisinhibitor, or both the necrosis inhibitor and the apoptosis inhibitorare administered sequentially or simultaneously.
 46. The method of claim1, wherein the necrosis inhibitor is a compound of Formula I:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein X is O or S; R₁ is hydrogen, C₁-C₆alkyl, C₁-C₆ alkoxyl, orhalogen; and R₂ is hydrogen or C₁-C₆alkyl.
 47. The method of claim 1,wherein the necrosis inhibitor is a compound of Formula I-A:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein R₁ is H, alkyl, alkoxyl, or a halogen; and R₂ is H or an alkyl.48. The method of claim 1, wherein the necrosis inhibitor is a compoundselected from the group consisting of a compound of Formula I-B:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof; acompound of Formula I-C:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof;and a compound of Formula I-D:

or a pharmaceutically acceptable salt thereof.
 49. The method of claim1, wherein the necrosis inhibitor is a compound of Formula I-E:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein R₁ is H, alkyl, alkoxyl, or a halogen; and R₂ is H or an alkyl.50. The method of claim 1, wherein the necrosis inhibitor is a compoundof Formula II:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: X is —CH₂—, —C(H)(R₁₄)—, —C(═S)—, —C(═NH)—, or —C(O)—; R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, and R₁₀ each represent independentlyhydrogen, acyl, acetyl, alkyl, halogen, amino, C₁-C₆alkoxyl, nitro,—C(O)R₁₂, —C(S)R₁₂, —C(O)OR₁₂, —C(O)NR₁₂R₁₃, —C(S)NR₁₂R₁₃, or —S(O₂)R₁₂;R₁₁ is hydrogen, acyl, acetyl, alkyl, or acylamino; R₁₂ and R₁₃ eachrepresent independently hydrogen, an optionally substituted alkyl, anoptionally substituted aryl, an optionally substituted heteroaryl, anoptionally substituted aralkyl, or an optionally substitutedheteroaralkyl; R₁₄ is acyl, acetyl, alkyl, halogen, amino, acylamino,nitro, —SR₁₁, —N(R₁₁)₂, or —OR₁₁; the bond indicated by (a) can be asingle bond or a double bond; and the bond indicated by (b) can be asingle bond or a double bond.
 51. The method of claim 1, wherein thenecrosis inhibitor is a compound of Formula II-A:

or a pharmaceutically acceptable salt thereof, wherein: R₁, R₂, R₅, R₆,R₇, and R₁₀ each represent independently hydrogen, alkyl, halogen,amino, or methoxyl; and R₃, R₄, R₈, and R₉ are C₁-C₆alkoxyl.
 52. Themethod of claim 1, wherein the necrosis inhibitor is a compound ofFormula III:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: Z is —CH₂—, —CH₂CH₂—, —O—, —S—, —S(O)—, —S(O₂)—, or —N(R₇)—;R₁, R₃, and R₅ each represent independently for each occurrencehydrogen, halogen, hydroxyl, amino, C₁-C₆alkyl, C₁-C₆alkoxy,C₁-C₆alkoxy-C₁-C₆alkyl, C₁-C₆alkanoyl, C₁-C₆alkylsulfinyl,C₁-C₆alkylsulfinyl-C₁-C₆alkyl, C₁-C₆alkylsulfonyl,C₁-C₆alkylsulfonyl-C₁-C₆alkyl, aryl, aralkyl, heterocycloalkyl,heteroaryl, or heteroaralkyl; R₂ and R₄ are C₁-C₆alkoxy; R₆ is —C(O)R₈,—C(S)R₈, —C(O)OR₈, —C(O)NR₈R₉, —C(S)NR₈R₉, —C(NH)R₈, or —S(O₂)R₈; R₇ isalkyl, aralkyl, or heteroaralkyl; R₈ and R₉ each represent independentlyhydrogen, C₁-C₆alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; and n represents independently for each occurrence 0, 1,or
 2. 53. The method of claim 1, wherein the necrosis inhibitor is acompound of Formula IV:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: R₁ is

R₂ and R₃ each represent independently for each occurrence hydrogen ormethyl; R₄ represents independently for each occurrence halogen,hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, or C₂-C₄alkynyl; R₅ is C₁-C₄alkyl;R₆ is hydrogen, halogen, or —CN; R₇ is hydrogen or C₁-C₄alkyl; R₈ isC₁-C₆alkyl, or R₈ taken together with R₉, when present, forms acarbocyclic ring; R₉ is hydrogen or C₁-C₆alkyl, or R₉ taken togetherwith R₈ forms a carbocyclic ring; R₁₀ is hydrogen or C₁-C₆alkyl; A isphenylene or a 5-6 membered heteroarylene; X is N or —C(R₉)—; Y is N or—C(R₁₀)—; Z is S or O; and m and n each represent independently 1, 2, or3.
 54. The method of claim 1, wherein the necrosis inhibitor is acompound of Formula V:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: A is a saturated or unsaturated 5-6 membered carbocyclic ring;X is a bond or C₁-C₄alkylene; R₁ is C₁-C₆ alkyl, halogen, hydroxyl,C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄, CO₂R₄, or C(O)N(R₄)₂; R₂ is

R₃ is —C₁-C₆alkylene-CN, —CN, C₁-C₆alkyl, or C₂-C₆alkenyl; R₄ representsindependently for each occurrence hydrogen, C₁-C₆alkyl, aryl, oraralkyl; R₅ represents independently for each occurrence C₁-C₆alkyl,halogen, hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂, —C(O)R₄, CO₂R₄, or C(O)N(R₄)₂;B is a 5-6 membered heterocyclic or carbocylic ring; and n and p eachrepresent independently 0, 1, or
 2. 55. The method of claim 1, whereinthe necrosis inhibitor is a compound of Formula V-A:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: R₁ is C₁-C₆alkyl, halogen, hydroxyl, C₁-C₆alkoxyl, or —N(R₄)₂;R₂ is

R₃ is —C₁-C₆alkylene-CN; R₄ represents independently for each occurrencehydrogen, C₁-C₆alkyl, aryl, or aralkyl; R₅ represents independently foreach occurrence C₁-C₆alkyl, halogen, hydroxyl, C₁-C₆alkoxyl, —N(R₄)₂,—C(O)R₄, CO₂R₄, or C(O)N(R₄)₂; B is a 5-6 membered heterocyclic orcarbocylic ring; and n and p each represent independently 0, 1, or 2.56. The method of claim 1, wherein the necrosis inhibitor is a compoundof Formula VII:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: R₁, R₂, and R₃ each represent independently hydrogen orC₁-C₄alkyl; R₄ is

R₅ and R₆ each represent independently for each occurrence halogen,C₁-C₆alkyl, hydroxyl, C₁-C₆alkoxyl, —N(R₇)₂, —NO₂, —S—C₁-C₆alkyl,—S-aryl, —SO₂—C₁-C₆alkyl, —SO₂-aryl, —C(O)R₇, —CO₂R₇, —C(O)N(R₇)₂,heterocycloalkyl, aryl, or heteroaryl; R₇ represents independently foreach occurrence hydrogen, C₁-C₆alkyl, aryl, or aralkyl; or twooccurrences of R₇ attached to the same nitrogen atom are taken togetherwith the nitrogen atom to which they are attached to form a 3-7 memberedheterocyclic ring; A is a 5-6 membered heterocyclic ring; and p is 0, 1,or
 2. 57. The method of claim 1, wherein the necrosis inhibitor is acompound of Formula VIII:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: each X¹, X², X³, X⁴, X⁵, and X⁶ is selected, independently,from N or CR^(X1); each Y¹, Y², and Y³ is selected, independently, fromO, S, NR^(Y1), or CR^(Y2)R^(Y3); each Z¹ and Z² is selected,independently, from O, S, or NR^(Z1); each R^(Y1) and R^(Z1) isselected, independently, from H, optionally substituted C₁-C₆alkyl,optionally substituted C₂-C₆alkenyl, optionally substitutedC₂-C₆alkynyl, optionally substituted cycloalkyl, optionally substitutedheterocyclyl, optionally substituted aryl, optionally substitutedheteroaryl, —C(═O)R^(5A), —C(═O)OR^(5A), or —C(═O)NR^(5A)R^(6A); eachR^(X1), R^(Y2), and R^(Y3) is selected, independently, from H, halogen,CN, NC, NO₂, N₃, OR³, SR³, NR³R⁴, —C(═O)R^(5A), —C(═O)OR^(5A),—C(═O)NR^(5A)R^(6A), —S(═O)R^(5A), —S(═O)₂OR^(5A), —S(═O)₂OR^(5A),—S(═O)₂OR^(5A)R^(6A), optionally substituted C₁-C₆alkyl, optionallysubstituted C₂-C₆alkenyl, optionally substituted C₂-C₆alkynyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl; eachR¹, R² R^(5A), R^(5B), R^(6A), and R^(6B) is selected from H, optionallysubstituted C₁-C₆ alkyl, optionally substituted C₂-C₆alkenyl, optionallysubstituted C₂-C₆alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl; or R^(5A) and R^(6A), or R^(5B) and R^(6B)combine to form a heterocyclyl; and each R³ and R⁴ is selected from H,optionally substituted C₁-C₆ alkyl, optionally substituted cycloalkyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, —C(═O)R^(5B), —C(═S)R^(5B),—C(═NR^(6B))R^(5B), —C(═O)OR^(5B), —C(═O)NR^(5B)R^(6B), —S(═O)R^(5B),—S(═O)₂R^(5B), —S(═O)₂OR^(5B), or —S(═O)₂NR^(5B)R^(6B).
 58. The methodof claim 1, wherein the necrosis inhibitor is a compound of Formula IX:

or a pharmaceutically acceptable salt, an ester, or a prodrug thereof,wherein: X₁ and X₂ are, independently, N or CR⁴; X₃ is selected from O,S, NR⁵, or (CR⁵)₂; Y is selected from C(O) or CH₂; and Z is (CR⁶R⁷)_(n);R¹ is selected from H, halogen, optionally substituted C₁-C₆alkyl,optionally substituted C₁-C₆cycloalkyl, or optionally substituted aryl;R² is selected from H or optionally substituted C₁-C₆alkyl; R³ isoptionally substituted aryl; each R⁴ is selected from H, halogen,carboxamido, nitro, cyano, optionally substituted C₁-C₆alkyl, oroptionally substituted aryl; R⁵ is selected from H, halogen, optionallysubstituted C₁-C₆alkyl, or optionally substituted aryl; each R⁶ and R⁷is, independently, selected from H, optionally substituted C₁-C₆alkyl,or aryl; and n is 0, 1, 2, or
 3. 59-64. (canceled)
 65. The method ofclaim 15, wherein the ischemic optic neuropathy is selected from thegroup consisting of arteritic anterior ischemic neuropathy,non-arteritic anterior ischemic neuropathy, and posterior ischemic opticneuropathy.
 66. The method of claim 15, wherein the mitochondrial opticneuropathy is Leber's optic neuropathy.
 67. The method of claim 15,wherein the hereditary optic neuropathy is selected from the groupconsisting of Leber's optic neuropathy, Dominant Optic Atrophy, andBehr's syndrome.